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Nicotinamide adenine dinucleotide (NAD) coenzymes are carriers of high energy electrons in metabolism and also play critical roles in numerous signaling pathways. NAD metabolism is decreased in various cardiovascular diseases. Importantly, stimulation of NAD biosynthesis protects against heart disease under different pathological conditions. In this review, we describe pathways for both generation and catabolism of NAD coenzymes and the respective changes of these pathways in the heart under cardiac diseases, including pressure overload, myocardial infarction, cardiometabolic disease, cancer treatment cardiotoxicity, and heart failure. We next provide an update on the strategies and treatments to increase NAD levels, such as supplementation of NAD precursors, in the heart that prevent or reverse cardiomyopathy. We also introduce the approaches to manipulate NAD consumption enzymes to ameliorate cardiac disease. Finally, we discuss the mechanisms associated with improvements in cardiac function by NAD coenzymes, differentiating between mitochondria-dependent effects and those independent of mitochondrial metabolism.

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Small extracellular vesicles are nanosized vesicles (30-200 nm) that can ferry proteins, nucleic acids, and lipids between cells and therefore, have significant potential as biomarkers, drug delivery tools or therapeutic agents. SEVs of endothelial origin have been shown to -among other functions-reduce in vitro ischemia/reperfusion (I/R) injury in cardiomyocytes, but whether a pro-inflammatory state of the endothelium impairs the functionality of these SEVs remains to be elucidated. To test this, human umbilical vein endothelial cells cells were treated with TNF-α 10 ng/mL and the expression of the pro-inflammatory parameters VCAM-1, ICAM-1 and eNOS were determined by Western blot. SEVs were isolated from endothelial cells treated with or without TNF-α 10 ng/mL using size exclusion chromatography. The size and concentration of SEVs was measured by Nanoparticle Tracking Analysis. The expression of the surface marker CD81 was determined by immunoassay, whereas their morphology was assessed by electron microscopy. The function of endothelial SEVs was assessed by evaluating their cardioprotective effect in an ex vivo model of global I/R using isolated hearts from adult C57BL/6 mice. Treatment of HUVECs with TNF-α induced the expression of VCAM-1 and ICAM-1, whereas eNOS levels were decreased. TNF-α did not affect the production, size, morphology, or expression of CD81. SEVs significantly reduced the infarct size as compared with untreated mice hearts, but SEVs isolated from TNF-α treated cells were unable to achieve this effect. Therefore, a pro-inflammatory state induced by TNF-α does not alter the production of endothelial SEVs but impairs their function in the setting of I/R injury.

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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.

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Heart failure with preserved ejection fraction (HFpEF) is one of the most complex and most prevalent cardiometabolic diseases in aging population. Age, obesity, diabetes, and hypertension are the main comorbidities of HFpEF. Microvascular dysfunction and vascular remodeling play a major role in its development. Among the many mechanisms involved in this process, vascular stiffening has been described as one the most prevalent during HFpEF, leading to ventricular-vascular uncoupling and mismatches in aged HFpEF patients. Aged blood vessels display an increased number of senescent endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). This is consistent with the fact that EC and cardiomyocyte cell senescence has been reported during HFpEF. Autophagy plays a major role in VSMCs physiology, regulating phenotypic switch between contractile and synthetic phenotypes. It has also been described that autophagy can regulate arterial stiffening and EC and VSMC senescence. Many studies now support the notion that targeting autophagy would help with the treatment of many cardiovascular and metabolic diseases. In this review, we discuss the mechanisms involved in autophagy-mediated vascular senescence and whether this could be a driver in the development and progression of HFpEF.

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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.

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Mitochondrial E3 ubiquitin ligase 1 (MUL1) is a mitochondrial outer membrane-anchored protein-containing transmembrane domain in its N- and C-terminal regions, where both are exposed to the cytosol. Interestingly the C-terminal region has a RING finger domain responsible for its E3 ligase activity, as ubiquitin or in SUMOylation, interacting with proteins related to mitochondrial fusion and fission, cell survival, and tumor suppressor process, such as Akt. Therefore, MUL1 is involved in various cellular processes, such as mitochondrial dynamics, inter-organelle communication, proliferation, mitophagy, immune response, inflammation and cell apoptosis. MUL1 is expressed at a higher basal level in the heart, immune system organs, and blood. Here, we discuss the role of MUL1 in mitochondrial dynamics and its function in various pathological models, both in vitro and in vivo. In this context, we describe the role of MUL1 in: (1) the inflammatory response, by regulating NF-κB activity; (2) cancer, by promoting cell death and regulating exonuclear function of proteins, such as p53; (3) neurological diseases, by maintaining communication with other organelles and interacting with proteins to eliminate damaged organelles and; (4) cardiovascular diseases, by maintaining mitochondrial fusion/fission homeostasis. In this review, we summarize the latest advances in the physiological and pathological functions of MUL1. We also describe the different substrates of MUL1, acting as a positive or negative regulator in various pathologies associated with mitochondrial dysfunction. In conclusion, MUL1 could be a potential key target for the development of therapies that focus on ensuring the functionality of the mitochondrial network and, furthermore, the quality control of intracellular components by synchronously modulating the activity of different cellular mechanisms involved in the aforementioned pathologies. This, in turn, will guide the development of targeted therapies.

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Hypertension is associated with high circulating angiotensin II (Ang II). We have reported that autophagy regulates Ang II-induced vascular smooth muscle cell (VSMC) hypertrophy, but the mechanism mediating this effect is still unknown. Therefore, we studied how Ang II regulates LC3 levels in VSMCs and whether Bag3, a co-chaperone known to regulate LC3 total levels, may be involved in the effects elicited by Ang II. A7r5 cell line or rat aortic smooth muscle cell (RASMC) primary culture were stimulated with Ang II 100 nM for 24 h and LC3 I, LC3 II and Bag3 protein levels were determined by Western blot. MAP1LC3B mRNA levels were assessed by RT-qPCR. Ang II increased MAP1LC3B mRNA levels and protein levels of LC3 I, LC3 II and total LC3 (LC3 I + LC3 II). Cycloheximide, but not actinomycin D, abolished LC3 II and total LC3 increase elicited by Ang II in RASMCs. In A7r5 cells, cycloheximide prevented the Ang II-mediated increase of LC3 I and total LC3, but not LC3 II. Moreover, Ang II increased Bag3 levels, but this increase was not observed upon co-administration with either losartan 1 µM (AT1R antagonist) or Y-27632 10 µM (ROCK inhibitor). These results suggest that Ang II may regulate total LC3 content through transcriptional and translational mechanisms. Moreover, Bag3 is increased in response to Ang II by a AT1R/ROCK signalling pathway. These data provide preliminary evidence suggesting that Ang II may stimulate autophagy in VSMCs by increasing total LC3 content and LC3 processing.

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Little is known about the effects of training load on exercise-induced plasma increase of interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) and their relationship with vascular remodeling. We sought to evaluate the role of sIL 6R as a regulator of IL-6-induced vascular remodeling. Forty-four male marathon runners were recruited and allocated into two groups: low-training (LT, <100 km/week) and high-training (HT, ≥100 km/week), 22 athletes per group. Twenty-one sedentary participants were used as reference. IL-6, sIL-6R and sgp130 levels were measured in plasma samples obtained before and immediately after finishing a marathon (42.2-km). Aortic diameter was measured by echocardiography. The inhibitory effect of sIL-6R on IL-6-induced VSMC migration was assessed using cultured A7r5 VSMCs. Basal plasma IL-6 and sIL-6R levels were similar among sedentary and athlete groups. Plasma IL-6 and sIL-6R levels were elevated after the marathon, and HT athletes had higher post-race plasma sIL-6R, but not IL-6, level than LT athletes. No changes in sgp130 plasma levels were found in LT and HT groups before and after running the marathon. Athletes had a more dilated ascending aorta and aortic root than sedentary participants with no differences between HT and LT athletes. However, a positive correlation between ascending aorta diameter and plasma IL-6 levels corrected by training load and years of training was observed. IL-6 could be responsible for aorta dilation because IL-6 stimulated VSMC migration in vitro, an effect that is inhibited by sIL-6R. However, IL-6 did not modify cell proliferation, collagen type I and contractile protein of VSMC. Our results suggest that exercise induces vascular remodeling. A possible association with IL-6 is proposed. Because sIL-6R inhibits IL-6-induced VSMC migration, a possible mechanism to regulate IL-6-dependent VSMC migration is also proposed.

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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.

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IL-6 is usually described as a pleiotropic cytokine produced in response to tissue injury or infection. As a pro-inflammatory cytokine, IL-6 activates innate and adaptative immune responses. IL-6 is released in the innate immune response by leukocytes as well as stromal cells upon pattern recognition receptor activation. IL-6 then recruits immune cells and triggers B and T cell response. Dysregulated IL-6 activity is associated with pathologies involving chronic inflammation and autoimmunity, including atherosclerosis. However, IL-6 is also produced and released under beneficial conditions, such as exercise, where IL-6 is associated with the anti-inflammatory and metabolic effects coupled with physical adaptation to intense training. Exercise-associated IL-6 acts on adipose tissue to induce lipogenesis and on arteries to induce adaptative vascular remodeling. These divergent actions could be explained by complex signaling networks. Classical IL-6 signaling involves a membrane-bound IL-6 receptor and glycoprotein 130 (gp130), while trans-signaling relies on a soluble version of IL-6R (sIL-6R) and membrane-bound gp130. Trans-signaling, but not the classical pathway, is regulated by soluble gp130. In this review, we discuss the similarities and differences in IL-6 cytokine and myokine signaling to explain the differential and opposite effects of this protein during inflammation and exercise, with a special focus on the vascular system.