Organelle Communication: Joined in Sickness and in Health.

Organelles are membrane-lined structures that compartmentalize subcellular biochemical functions. Therefore, interorganelle communication is crucial for cellular responses that require the coordination of such functions. Multiple principles govern interorganelle interactions, which arise from the complex nature of organelles: position, multilingualism, continuity, heterogeneity, proximity, and bidirectionality, among others. Given their importance, alterations in organelle communication have been linked to many diseases. Among the different types of contacts, endoplasmic reticulum mitochondria interactions are the best known; however, mounting evidence indicates that other organelles also have something to say in the pathophysiological conversation.

IGF-1 boosts mitochondrial function by a Ca2+ uptake-dependent mechanism in cultured human and rat cardiomyocytes.

A physiological increase in cardiac workload results in adaptive cardiac remodeling, characterized by increased oxidative metabolism and improvements in cardiac performance. Insulin-like growth factor-1 (IGF-1) has been identified as a critical regulator of physiological cardiac growth, but its precise role in cardiometabolic adaptations to physiological stress remains unresolved. Mitochondrial calcium (Ca2+) handling has been proposed to be required for sustaining key mitochondrial dehydrogenase activity and energy production during increased workload conditions, thus ensuring the adaptive cardiac response. We hypothesized that IGF-1 enhances mitochondrial energy production through a Ca2+-dependent mechanism to ensure adaptive cardiomyocyte growth. We found that stimulation with IGF-1 resulted in increased mitochondrial Ca2+ uptake in neonatal rat ventricular myocytes and human embryonic stem cell-derived cardiomyocytes, estimated by fluorescence microscopy and indirectly by a reduction in the pyruvate dehydrogenase phosphorylation. We showed that IGF-1 modulated the expression of mitochondrial Ca2+ uniporter (MCU) complex subunits and increased the mitochondrial membrane potential; consistent with higher MCU-mediated Ca2+ transport. Finally, we showed that IGF-1 improved mitochondrial respiration through a mechanism dependent on MCU-mediated Ca2+ transport. In conclusion, IGF-1-induced mitochondrial Ca2+ uptake is required to boost oxidative metabolism during cardiomyocyte adaptive growth.

Effects of Trimetazidine on Right Ventricular Function and Ventricular Remodeling in Patients with Pulmonary Artery Hypertension: A Randomised Controlled Trial.

BACKGROUND: Pulmonary artery hypertension (PAH) is a chronic and progressive disease. Although current therapy has improved the disease prognosis, PAH has a poor survival rate. The key feature leading to disease progression and death is right ventricular (RV) failure. METHODS AND RESULTS: We assessed the role of trimetazidine, a fatty acid beta-oxidation (FAO) inhibitor, in right ventricular function, remodeling, and functional class in PAH patients, with a placebo-controlled double-blind, case-crossover trial. Twenty-seven PAH subjects were enrolled, randomized, and assigned to trimetazidine or placebo for three months and then reallocated to the other study arm. The primary endpoint was RV morphology and function change after three months of treatment. Secondary endpoints were the change in exercise capacity assessed by a 6 min walk test after three months of treatment and the change in pro-BNP and Galectin-3 plasma levels after three months. Trimetazidine use was safe and well-tolerated. After three months of treatment, patients in the trimetazidine group showed a small but significant reduction of RV diastolic area, and a substantial increase in the 6 min walk distance (418 vs. 438 mt, p = 0.023), without significant changes in biomarkers. CONCLUSIONS: A short course of trimetazidine is safe and well-tolerated on PAH patients, and it is associated with significant increases in the 6MWT and minor but significant improvement in RV remodeling. The therapeutic potential of this drug should be evaluated in larger clinical trials.

Angiotensin-(1-9) in hypertension.

Angiotensin-(1-9) [Ang-(1-9)] is a peptide of the non-canonical renin-angiotensin system (RAS) synthesized from angiotensin I by the monopeptidase angiotensin-converting enzyme type 2 (ACE2). Using osmotic minipumps, infusion of Ang-(1-9) consistently reduces blood pressure in several rat hypertension models. In these animals, hypertension-induced end-organ damage is also decreased. Several pieces of evidence suggest that Ang-(1-9) is the endogenous ligand that binds and activates the type-2 angiotensin II receptor (AT2R). Activation of AT2R triggers different tissue-specific signaling pathways. This phenomenon could be explained by the ability of AT2R to form different heterodimers with other G protein-coupled receptors. Because of the antihypertensive and protective effects of AT2R activation by Ang-(1-9), associated with a short half-life of RAS peptides, several synthetic AT2R agonists have been synthesized and assayed. Some of them, particularly CGP42112, C21 and novokinin, have demonstrated antihypertensive properties. Only two synthetic AT2R agonists, C21 and LP2-3, have been tested in clinical trials, but none of them like an antihypertensive. Therefore, Ang-(1-9) is a promising antihypertensive drug that reduces hypertension-induced end-organ damage. However, further research is required to translate this finding successfully to the clinic.

Polycystin-1 is required for insulin-like growth factor 1-induced cardiomyocyte hypertrophy.

Cardiac hypertrophy is the result of responses to various physiological or pathological stimuli. Recently, we showed that polycystin-1 participates in cardiomyocyte hypertrophy elicited by pressure overload and mechanical stress. Interestingly, polycystin-1 knockdown does not affect phenylephrine-induced cardiomyocyte hypertrophy, suggesting that the effects of polycystin-1 are stimulus-dependent. In this study, we aimed to identify the role of polycystin-1 in insulin-like growth factor-1 (IGF-1) signaling in cardiomyocytes. Polycystin-1 knockdown completely blunted IGF-1-induced cardiomyocyte hypertrophy. We then investigated the molecular mechanism underlying this result. We found that polycystin-1 silencing impaired the activation of the IGF-1 receptor, Akt, and ERK1/2 elicited by IGF-1. Remarkably, IGF-1-induced IGF-1 receptor, Akt, and ERK1/2 phosphorylations were restored when protein tyrosine phosphatase 1B was inhibited, suggesting that polycystin-1 knockdown deregulates this phosphatase in cardiomyocytes. Moreover, protein tyrosine phosphatase 1B inhibition also restored IGF-1-dependent cardiomyocyte hypertrophy in polycystin-1-deficient cells. Our findings provide the first evidence that polycystin-1 regulates IGF-1-induced cardiomyocyte hypertrophy through a mechanism involving protein tyrosine phosphatase 1B.

Polycystin-1 regulates cardiomyocyte mitophagy.

Polycystin-1 (PC1) is a transmembrane protein found in different cell types, including cardiomyocytes. Alterations in PC1 expression have been linked to mitochondrial damage in renal tubule cells and in patients with autosomal dominant polycystic kidney disease. However, to date, the regulatory role of PC1 in cardiomyocyte mitochondria is not well understood. The analysis of mitochondrial morphology from cardiomyocytes of heterozygous PC1 mice (PDK1+/- ) using transmission electron microscopy showed that cardiomyocyte mitochondria were smaller with increased mitochondria density and circularity. These parameters were consistent with mitochondrial fission. We knocked-down PC1 in cultured rat cardiomyocytes and human-induced pluripotent stem cells (iPSC)-derived cardiomyocytes to evaluate mitochondrial function and morphology. The results showed that downregulation of PC1 expression results in reduced protein levels of sub-units of the OXPHOS complexes and less functional mitochondria (reduction of mitochondrial membrane potential, mitochondrial respiration, and ATP production). This mitochondrial dysfunction activates the elimination of defective mitochondria by mitophagy, assessed by an increase of autophagosome adapter protein LC3B and the recruitment of the Parkin protein to the mitochondria. siRNA-mediated PC1 knockdown leads to a loss of the connectivity of the mitochondrial network and a greater number of mitochondria per cell, but of smaller sizes, which characterizes mitochondrial fission. PC1 silencing also deregulates the AKT-FoxO1 signaling pathway, which is involved in the regulation of mitochondrial metabolism, mitochondrial morphology, and processes that are part of cell quality control, such as mitophagy. Together, these data provide new insights about the controls that PC1 exerts on mitochondrial morphology and function in cultured cardiomyocytes dependent on the AKT-FoxO1 signaling pathway.

Endoplasmic reticulum-mitochondria coupling increases during doxycycline-induced mitochondrial stress in HeLa cells.

Subcellular organelles communicate with each other to regulate function and coordinate responses to changing cellular conditions. The physical-functional coupling of the endoplasmic reticulum (ER) with mitochondria allows for the direct transfer of Ca2+ between organelles and is an important avenue for rapidly increasing mitochondrial metabolic activity. As such, increasing ER-mitochondrial coupling can boost the generation of ATP that is needed to restore homeostasis in the face of cellular stress. The mitochondrial unfolded protein response (mtUPR) is activated by the accumulation of unfolded proteins in mitochondria. Retrograde signaling from mitochondria to the nucleus promotes mtUPR transcriptional responses aimed at restoring protein homeostasis. It is currently unknown whether the changes in mitochondrial-ER coupling also play a role during mtUPR stress. We hypothesized that mitochondrial stress favors an expansion of functional contacts between mitochondria and ER, thereby increasing mitochondrial metabolism as part of a protective response. Hela cells were treated with doxycycline, an antibiotic that inhibits the translation of mitochondrial-encoded proteins to create protein disequilibrium. Treatment with doxycycline decreased the abundance of mitochondrial encoded proteins while increasing expression of CHOP, C/EBPß, ClpP, and mtHsp60, markers of the mtUPR. There was no change in either mitophagic activity or cell viability. Furthermore, ER UPR was not activated, suggesting focused activation of the mtUPR. Within 2 h of doxycycline treatment, there was a significant increase in physical contacts between mitochondria and ER that was distributed throughout the cell, along with an increase in the kinetics of mitochondrial Ca2+ uptake. This was followed by the rise in the rate of oxygen consumption at 4 h, indicating a boost in mitochondrial metabolic activity. In conclusion, an early phase of the response to doxycycline-induced mitochondrial stress is an increase in mitochondrial-ER coupling that potentiates mitochondrial metabolic activity as a means to support subsequent steps in the mtUPR pathway and sustain cellular adaptation.

Novel molecular insights and public omics data in pulmonary hypertension.

Pulmonary hypertension is a rare disease with high morbidity and mortality which mainly affects women of reproductive age. Despite recent advances in understanding the pathogenesis of pulmonary hypertension, the high heterogeneity in the presentation of the disease among different patients makes it difficult to make an accurate diagnosis and to apply this knowledge to effective treatments. Therefore, new studies are required to focus on translational and personalized medicine to overcome the lack of specificity and efficacy of current management. Here, we review the majority of public databases storing 'omics' data of pulmonary hypertension studies, from animal models to human patients. Moreover, we review some of the new molecular mechanisms involved in the pathogenesis of pulmonary hypertension, including non-coding RNAs and the application of 'omics' data to understand this pathology, hoping that these new approaches will provide insights to guide the way to personalized diagnosis and treatment.

Novel Insights Into the Pathogenesis of Diabetic Cardiomyopathy and Pharmacological Strategies.

Diabetic cardiomyopathy (DCM) is a severe complication of diabetes developed mainly in poorly controlled patients. In DCM, several clinical manifestations as well as cellular and molecular mechanisms contribute to its phenotype. The production of reactive oxygen species (ROS), chronic low-grade inflammation, mitochondrial dysfunction, autophagic flux inhibition, altered metabolism, dysfunctional insulin signaling, cardiomyocyte hypertrophy, cardiac fibrosis, and increased myocardial cell death are described as the cardinal features involved in the genesis and development of DCM. However, many of these features can be associated with broader cellular processes such as inflammatory signaling, mitochondrial alterations, and autophagic flux inhibition. In this review, these mechanisms are critically discussed, highlighting the latest evidence and their contribution to the pathogenesis of DCM and their potential as pharmacological targets.

Angiotensin-(1-9) prevents cardiomyocyte hypertrophy by controlling mitochondrial dynamics via miR-129-3p/PKIA pathway.

Angiotensin-(1-9) is a peptide from the noncanonical renin-angiotensin system with anti-hypertrophic effects in cardiomyocytes via an unknown mechanism. In the present study we aimed to elucidate it, basing us initially on previous work from our group and colleagues who proved a relationship between disturbances in mitochondrial morphology and calcium handling, associated with the setting of cardiac hypertrophy. Our first finding was that angiotensin-(1-9) can induce mitochondrial fusion through DRP1 phosphorylation. Secondly, angiotensin-(1-9) blocked mitochondrial fission and intracellular calcium dysregulation in a model of norepinephrine-induced cardiomyocyte hypertrophy, preventing the activation of the calcineurin/NFAT signaling pathway. To further investigate angiotensin-(1-9) anti-hypertrophic mechanism, we performed RNA-seq studies, identifying the upregulation of miR-129 under angiotensin-(1-9) treatment. miR-129 decreased the transcript levels of the protein kinase A inhibitor (PKIA), resulting in the activation of the protein kinase A (PKA) signaling pathway. Finally, we showed that PKA activity is necessary for the effects of angiotensin-(1-9) over mitochondrial dynamics, calcium handling and its anti-hypertrophic effects.

Autophagy Activation in Zebrafish Heart Regeneration.

Autophagy is an evolutionarily conserved process that plays a key role in the maintenance of overall cellular health. While it has been suggested that autophagy may elicit cardioprotective and pro-survival modulating functions, excessive activation of autophagy can also be detrimental. In this regard, the zebrafish is considered a hallmark model for vertebrate regeneration, since contrary to adult mammals, it is able to faithfully regenerate cardiac tissue. Interestingly, the role that autophagy may play in zebrafish heart regeneration has not been studied yet. In the present work, we hypothesize that, in the context of a well-established injury model of ventricular apex resection, autophagy plays a critical role during cardiac regeneration and its regulation can directly affect the zebrafish regenerative potential. We studied the autophagy events occurring upon injury using electron microscopy, in vivo tracking of autophagy markers, and protein analysis. Additionally, using pharmacological tools, we investigated how rapamycin, an inducer of autophagy, affects regeneration relevant processes. Our results show that a tightly regulated autophagic response is triggered upon injury and during the early stages of the regeneration process. Furthermore, treatment with rapamycin caused an impairment in the cardiac regeneration outcome. These findings are reminiscent of the pathophysiological description of an injured human heart and hence put forward the zebrafish as a model to study the poorly understood double-sword effect that autophagy has in cardiac homeostasis.

Inhibition of the proteasome preserves Mitofusin-2 and mitochondrial integrity, protecting cardiomyocytes during ischemia-reperfusion injury.

Cardiomyocyte loss is the main cause of myocardial dysfunction following an ischemia-reperfusion (IR) injury. Mitochondrial dysfunction and altered mitochondrial network dynamics play central roles in cardiomyocyte death. Proteasome inhibition is cardioprotective in the setting of IR; however, the mechanisms underlying this protection are not well-understood. Several proteins that regulate mitochondrial dynamics and energy metabolism, including Mitofusin-2 (Mfn2), are degraded by the proteasome. The aim of this study was to evaluate whether proteasome inhibition can protect cardiomyocytes from IR damage by maintaining Mfn2 levels and preserving mitochondrial network integrity. Using ex vivo Langendorff-perfused rat hearts and in vitro neonatal rat ventricular myocytes, we showed that the proteasome inhibitor MG132 reduced IR-induced cardiomyocyte death. Moreover, MG132 preserved mitochondrial mass, prevented mitochondrial network fragmentation, and abolished IR-induced reductions in Mfn2 levels in heart tissue and cultured cardiomyocytes. Interestingly, Mfn2 overexpression also prevented cardiomyocyte death. This effect was apparently specific to Mfn2, as overexpression of Miro1, another protein implicated in mitochondrial dynamics, did not confer the same protection. Our results suggest that proteasome inhibition protects cardiomyocytes from IR damage. This effect could be partly mediated by preservation of Mfn2 and therefore mitochondrial integrity.

GDF-11 prevents cardiomyocyte hypertrophy by maintaining the sarcoplasmic reticulum-mitochondria communication.

Growth differentiation factor 11 (GDF11) is a novel factor with controversial effects on cardiac hypertrophy both in vivo and in vitro. Although recent evidence has corroborated that GDF11 prevents the development of cardiac hypertrophy, its molecular mechanism remains unclear. In our previous work, we showed that norepinephrine (NE), a physiological pro-hypertrophic agent, increases cytoplasmic Ca2+ levels accompanied by a loss of physical and functional communication between sarcoplasmic reticulum (SR) and mitochondria, with a subsequent reduction in the mitochondrial Ca2+ uptake and mitochondrial metabolism. In order to study the anti-hypertrophic mechanism of GDF11, our aim was to investigate whether GDF11 prevents the loss of SR-mitochondria communication triggered by NE. Our results show that: a) GDF11 prevents hypertrophy in cultured neonatal rat ventricular myocytes treated with NE. b) GDF11 attenuates the NE-induced loss of contact sites between both organelles. c) GDF11 increases oxidative mitochondrial metabolism by stimulating mitochondrial Ca2+ uptake. In conclusion, the GDF11-dependent maintenance of physical and functional communication between SR and mitochondria is critical to allow Ca2+ transfer between both organelles and energy metabolism in the cardiomyocyte and to avoid the activation of Ca2+-dependent pro-hypertrophic signaling pathways.

Author Correction: Caveolin-1 impairs PKA-DRP1-mediated remodelling of ER-mitochondria communication during the early phase of ER stress.

Following publication of the article, the authors wrote to point out the following mistake.

Caveolin-1 impairs PKA-DRP1-mediated remodelling of ER-mitochondria communication during the early phase of ER stress.

Close contacts between endoplasmic reticulum and mitochondria enable reciprocal Ca2+ exchange, a key mechanism in the regulation of mitochondrial bioenergetics. During the early phase of endoplasmic reticulum stress, this inter-organellar communication increases as an adaptive mechanism to ensure cell survival. The signalling pathways governing this response, however, have not been characterized. Here we show that caveolin-1 localizes to the endoplasmic reticulum-mitochondria interface, where it impairs the remodelling of endoplasmic reticulum-mitochondria contacts, quenching Ca2+ transfer and rendering mitochondrial bioenergetics unresponsive to endoplasmic reticulum stress. Protein kinase A, in contrast, promotes endoplasmic reticulum and mitochondria remodelling and communication during endoplasmic reticulum stress to promote organelle dynamics and Ca2+ transfer as well as enhance mitochondrial bioenergetics during the adaptive response. Importantly, caveolin-1 expression reduces protein kinase A signalling, as evidenced by impaired phosphorylation and alterations in organelle distribution of the GTPase dynamin-related protein 1, thereby enhancing cell death in response to endoplasmic reticulum stress. In conclusion, caveolin-1 precludes stress-induced protein kinase A-dependent remodelling of endoplasmic reticulum-mitochondria communication.

Sarcoplasmic reticulum-mitochondria communication in cardiovascular pathophysiology.

Repetitive, calcium-mediated contractile activity renders cardiomyocytes critically dependent on a sustained energy supply and adequate calcium buffering, both of which are provided by mitochondria. Moreover, in vascular smooth muscle cells, mitochondrial metabolism modulates cell growth and proliferation, whereas cytosolic calcium levels regulate the arterial vascular tone. Physical and functional communication between mitochondria and sarco/endoplasmic reticulum and balanced mitochondrial dynamics seem to have a critical role for optimal calcium transfer to mitochondria, which is crucial in calcium homeostasis and mitochondrial metabolism in both types of muscle cells. Moreover, mitochondrial dysfunction has been associated with myocardial damage and dysregulation of vascular smooth muscle proliferation. Therefore, sarco/endoplasmic reticulum-mitochondria coupling and mitochondrial dynamics are now viewed as relevant factors in the pathogenesis of cardiac and vascular diseases, including coronary artery disease, heart failure, and pulmonary arterial hypertension. In this Review, we summarize the evidence related to the role of sarco/endoplasmic reticulum-mitochondria communication in cardiac and vascular muscle physiology, with a focus on how perturbations contribute to the pathogenesis of cardiovascular disorders.

Calcium Transport and Signaling in Mitochondria.

Calcium (Ca2+) is a key player in the regulation of many cell functions. Just like Ca2+, mitochondria are ubiquitous, versatile, and dynamic players in determining both cell survival and death decisions. Given their ubiquitous nature, the regulation of both is deeply intertwined, whereby Ca2+ regulates mitochondrial functions, while mitochondria shape Ca2+ dynamics. Deregulation of either Ca2+ or mitochondrial signaling leads to abnormal function, cell damage or even cell death, thereby contributing to muscle dysfunction or cardiac pathologies. Moreover, altered mitochondrial Ca2+ homeostasis has been linked to metabolic diseases like cancer, obesity, and pulmonary hypertension. In this review article, we summarize the mechanisms that coordinate mitochondrial and Ca2+ responses and how they affect human health. © 2017 American Physiological Society. Compr Physiol 7:623-634, 2017.

mTORC1 inhibitor rapamycin and ER stressor tunicamycin induce differential patterns of ER-mitochondria coupling.

Efficient mitochondrial Ca2+ uptake takes place at contact points between the ER and mitochondria, and represents a key regulator of many cell functions. In a previous study with HeLa cells, we showed that ER-to-mitochondria Ca2+ transfer increases during the early phase of ER stress induced by tunicamycin as an adaptive response to stimulate mitochondrial bioenergetics. It remains unknown whether other types of stress signals trigger similar responses. Here we observed that rapamycin, which inhibits the nutrient-sensing complex mTORC1, increased ER-mitochondria coupling in HeLa cells to a similar extent as did tunicamycin. Interestingly, although global responses to both stressors were comparable, there were notable differences in the spatial distribution of such changes. While tunicamycin increased organelle proximity primarily in the perinuclear region, rapamycin increased organelle contacts throughout the entire cell. These differences were paralleled by dissimilar alterations in the distribution of regulatory proteins of the ER-mitochondria interface, heterogeneities in mitochondrial Ca2+ uptake, and the formation of domains within the mitochondrial network with varying mitochondrial transmembrane potential. Collectively, these data suggest that while increasing ER-mitochondria coupling appears to represent a general response to cell stress, the intracellular distribution of the associated responses needs to be tailored to meet specific cellular requirements.

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism.

Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells.

FK866 compromises mitochondrial metabolism and adaptive stress responses in cultured cardiomyocytes.

AIM: FK866 is an inhibitor of the NAD(+) synthesis rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT). Using FK866 to target NAD(+) synthesis has been proposed as a treatment for inflammatory diseases and cancer. However, use of FK866 may pose cardiovascular risks, as NAMPT expression is decreased in various cardiomyopathies, with low NAD(+) levels playing an important role in cardiovascular disease progression. In addition, low NAD(+) levels are associated with cardiovascular risk conditions such as aging, dyslipidemia, and type II diabetes mellitus. The aim of this work was to study the effects of FK866-induced NAD(+) depletion on mitochondrial metabolism and adaptive stress responses in cardiomyocytes. METHODS AND RESULTS: FK866 was used to deplete NAD(+) levels in cultured rat cardiomyocytes. Cell viability, mitochondrial metabolism, and adaptive responses to insulin, norepinephrine, and H2O2 were assessed in cardiomyocytes. The drop in NAD(+) induced by FK866 decreased mitochondrial metabolism without changing cell viability. Insulin-stimulated Akt phosphorylation, glucose uptake, and H2O2-survival were compromised by FK866. Glycolytic gene transcription was increased, whereas cardiomyocyte hypertrophy induced by norepinephrine was prevented. Restoring NAD(+) levels via nicotinamide mononucleotide administration reestablished mitochondrial metabolism and adaptive stress responses. CONCLUSION: This work shows that FK866 compromises mitochondrial metabolism and the adaptive response of cardiomyocytes to norepinephrine, H2O2, and insulin.