Jaime A. Riquelme

Drp1 loss-of-function reduces cardiomyocyte oxygen dependence protecting the heart from ischemia-reperfusion injury.

Abstract: Mitochondria are key organelles for ATP production in cardiomyocytes, which is regulated by processes of fission and fusion. We hypothesized that the mitochondria fusion protein dynamin-related protein 1 (Drp1) inhibition, attenuates ischemia-reperfusion (I/R) injury through modifications in mitochondrial metabolism. Rats were subjected to I/R through coronary artery ligation, and isolated cardiomyocytes were treated with an ischemia-mimicking solution. In vivo, cardiac function, myocardial infarction area, and mitochondrial morphology were determined, whereas in vitro, viability, mitochondrial membrane potential, intracellular ATP levels, and oxygen consumption rate (OCR) were assessed. In both models, an adenovirus expressing Drp1 dominant-negative K38A (Drp1K38A) was used to induce Drp1 loss-of-function. Our results showed that I/R stimulated mitochondrial fission. Myocardial infarction size and cell death induced by I/R were significantly reduced, whereas cardiac function after I/R was improved in Drp1K38A-treated rats compared with controls. Drp1K38A-transduced cardiomyocytes showed lower OCR with no decrease in intracellular ATP levels, and on I/R, a larger decrease in OCR with a smaller reduction in intracellular ATP level was observed. However, proton leak-associated oxygen consumption was comparatively higher in Drp1K38A-treated cardiomyocytes, suggesting a protective mitochondrial uncoupling effect against I/R. Collectively, our results show that Drp1 inhibition triggers cardioprotection by reducing mitochondrial metabolism during I/R.

New Molecular Insights of Insulin in Diabetic Cardiomyopathy

Type 2 diabetes mellitus (T2DM) is a highly prevalent disease worldwide. Cardiovascular disorders generated as a consequence of T2DM are a major cause of death related to this disease. Diabetic cardiomyopathy (DCM) is characterized by the morphological, functional and metabolic changes in the heart produced as a complication of T2DM. This cardiac disorder is characterized by constant high blood glucose and lipids levels which eventually generate oxidative stress, defective calcium handling, altered mitochondrial function, inflammation and fibrosis. In this context, insulin is of paramount importance for cardiac contractility, growth and metabolism and therefore, an impaired insulin signaling plays a critical role in the DCM development. However, the exact pathophysiological mechanisms leading to DCM are still a matter of study. Despite the numerous questions raised in the study of DCM, there have also been important findings, such as the role of micro-RNAs (miRNAs), which can not only have the potential of being important biomarkers, but also therapeutic targets. Furthermore, exosomes also arise as an interesting variable to consider, since they represent an important inter-cellular communication mechanism and therefore, they may explain many aspects of the pathophysiology of DCM and their study may lead to the development of therapeutic agents capable of improving insulin signaling. In addition, adenosine and adenosine receptors (ARs) may also play an important role in DCM. Moreover, the possible cross-talk between insulin and ARs may provide new strategies to reverse its defective signaling in the diabetic heart. This review focuses on DCM, the role of insulin in this pathology and the discussion of new molecular insights which may help to understand its underlying mechanisms and generate possible new therapeutic strategies.

Regulation of cardiac autophagy by insulin-like growth factor 1

Insulin‐like growth factor‐1 (IGF‐1) signaling is a key pathway in the control of cell growth and survival. Three critical nodes in the IGF‐1 signaling pathway have been described in cardiomyocytes: protein kinase Akt/mammalian target of rapamycin (mTOR), Ras/Raf/extracellular signal‐regulated kinase (ERK), and phospholipase C (PLC)/inositol 1,4,5‐triphosphate (InsP3)/Ca2+. The Akt/mTOR and Ras/Raf/ERK signaling arms govern survival in the settings of cardiac stress and hypertrophic growth. By contrast, PLC/InsP3/Ca2+ functions to regulate metabolic adaptability and gene transcription. Autophagy is a catabolic process involved in protein degradation, organelle turnover, and nonselective breakdown of cytoplasmic components during nutrient starvation or stress. In the heart, autophagy is observed in a variety of human pathologies, where it can be either adaptive or maladaptive, depending on the context. We proposed the hypothesis that IGF‐1 protects the heart by rescuing the mitochondrial metabolism and the energetics state, reducing cell death and controls the potentially exacerbate autophagic response to nutritional stress. In light of the importance of IGF‐1 and autophagy in the heart, we review here IGF‐1 signaling and autophagy regulation in the context of cardiomyocyte nutritional stress.

Dexmedetomidine protects the heart against ischemia-reperfusion injury by an endothelial eNOS/NO dependent mechanism.

The alpha2-adrenergic receptor agonist Dexmedetomidine (Dex) is a sedative medication used by anesthesiologists. Dex protects the heart against ischemia-reperfusion (IR) and can also act as a preconditioning mimetic. The mechanisms involved in Dex-dependent cardiac preconditioning, and whether this action occurs directly or indirectly on cardiomyocytes, still remain unclear. The endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway and endothelial cells are known to play key roles in cardioprotection against IR injury. Therefore, the aims of this work were to evaluate whether the eNOS/NO pathway mediates the pharmacological cardiac effect of Dex, and whether endothelial cells are required in this cardioprotective action. Isolated adult rat hearts were treated with Dex (10nM) for 25min and the dimerization of eNOS and production of NO were measured. Hearts were then subjected to global IR (30/120min) and the role of the eNOS/NO pathway was evaluated. Dex promoted the activation of eNOS and production of NO. Dex reduced the infarct size and improved the left ventricle function recovery, but this effect was reversed when Dex was co-administered with inhibitors of the eNOS/NO/PKG pathway. In addition, Dex was unable to reduce cell death in isolated adult rat cardiomyocytes subjected to simulated IR. Cardiomyocyte death was attenuated by co-culturing them with endothelial cells pre-treated with Dex. In summary, our results show that Dex triggers cardiac protection by activating the eNOS/NO signaling pathway. This pharmacological effect of Dex requires its interaction with the endothelium.

Therapeutic targeting of autophagy in myocardial infarction and heart failure

ABSTRACT Introduction: Myocardial infarction (MI) is the leading cause of death. When MI is not lethal, heart failure (HF) is a major consequence with high prevalence and poor prognosis. The targeting of autophagy represents a potentially therapeutic approach for the treatment of both pathologies. Areas covered: PubMed searches were performed to discuss the current state of the art regarding the role of autophagy in MI and HF. We review available and potential approaches to modulate autophagy from a pharmacological and genetic perspective. We also discuss the targeting of autophagy in myocardial regeneration. Expert commentary: The targeting of autophagy has potential for the treatment of MI and HF. Autophagy is a process that takes place in virtually all cells of the body and thus, in order to evaluate this therapeutic approach in clinical trials, strategies that specifically target this process in the myocardium is required to avoid unwanted effects in other organs.

Role of PI3K in myocardial ischaemic preconditioning: mapping pro-survival cascades at the trigger phase and at reperfusion

The Reperfusion Injury Salvage Kinase (RISK) pathway is considered the main pro‐survival kinase cascade mediating the ischaemic preconditioning (IPC) cardioprotective effect. To assess the role of PI3K‐Akt, its negative regulator PTEN and other pro‐survival proteins such as ERK and STAT3 in the context of IPC, C57BL/6 mouse hearts were retrogradely perfused in a Langendorff system and subjected to 4 cycles of 5 min. ischaemia and 5 min. reperfusion prior to 35 min. of global ischaemia and 120 min. of reperfusion. Wortmannin, a PI3K inhibitor, was administered either at the stabilization period or during reperfusion. Infarct size was assessed using triphenyl tetrazolium staining, and phosphorylation levels of Akt, PTEN, ERK, GSK3β and STAT3 were evaluated using Western blot analyses. IPC reduced infarct size in hearts subjected to lethal ischaemia and reperfusion, but this effect was lost in the presence of Wortmannin, whether it was present only during preconditioning or only during early reperfusion. IPC increased the levels of Akt phosphorylation during both phases and this effect was fully abrogated by PI3K, whilst its downstream GSK3β was phosphorylated only during the trigger phase after IPC. Both PTEN and STAT3 were phosphorylated during both phases after IPC, but this was PI3K independent. IPC increases ERK phosphorylation during both phases, being only PI3K‐dependent during the IPC phase. In conclusion, PI3K‐Akt plays a major role in IPC‐induced cardioprotection. However, PTEN, ERK and STAT3 are also phosphorylated by IPC through a PI3K‐independent pathway, suggesting that cardioprotection is mediated through more than one cell signalling cascade.

Novel Therapies Targeting Cardioprotection and Regeneration.

Cardiovascular disease is the leading cause of death worldwide. The heart is susceptible to pathologies that impact the myocardium directly, such as myocardial infarction and consequent heart failure, as well as conditions with indirect cardiac effects, such as cancer treatment-related cardiotoxicity. As the contractile cells of the heart, cardiomyocytes are essential for normal cardiac function. Various stress stimuli may result in transient damage or cell death in cardiomyocytes through apoptosis, necrosis or maladaptive autophagy. Moreover, cardiomyocytes are unable to regenerate; thus, lost cells are replaced with fibrotic tissue, with a potentially severe impact on myocardial function. Several therapeutic agents and strategies to reduce cardiomyocyte damage are currently available. This manuscript reviews the state of the art regarding novel cardioprotective endogenous peptides, such as neuregulin-1, angiotensin-(1-9), growth/differentiation factor-11, growth/differentiation factor- 15 and insulin-like growth factor-1. We discuss their protective effects and therapeutic potential in cardiovascular diseases and the current challenges to harnessing their full cardioprotective power. We also explore targeting of exosomes as a cardioprotective approach along with the therapeutic potential of cardiac regeneration strategies. Further advances associated with these molecules and cardioprotective approaches may provide more effective therapies to attenuate or prevent cardiomyocyte death, thereby preserving the myocardium.

High-Fat-Diet-Induced Obesity Produces Spontaneous Ventricular Arrhythmias and Increases the Activity of Ryanodine Receptors in Mice

Ventricular arrhythmias are a common cause of sudden cardiac death, and their occurrence is higher in obese subjects. Abnormal gating of ryanodine receptors (RyR2), the calcium release channels of the sarcoplasmic reticulum, can produce ventricular arrhythmias. Since obesity promotes oxidative stress and RyR2 are redox-sensitive channels, we investigated whether the RyR2 activity was altered in obese mice. Mice fed a high fat diet (HFD) became obese after eight weeks and exhibited a significant increase in the occurrence of ventricular arrhythmias. Single RyR2 channels isolated from the hearts of obese mice were more active in planar bilayers than those isolated from the hearts of the control mice. At the molecular level, RyR2 channels from HFD-fed mice had substantially fewer free thiol residues, suggesting that redox modifications were responsible for the higher activity. Apocynin, provided in the drinking water, completely prevented the appearance of ventricular arrhythmias in HFD-fed mice, and normalized the activity and content of the free thiol residues of the protein. HFD increased the expression of NOX4, an isoform of NADPH oxidase, in the heart. Our results suggest that HFD increases the activity of RyR2 channels via a redox-dependent mechanism, favoring the appearance of ventricular arrhythmias.

Activation of Chymotrypsin-Like Activity of the Proteasome during Ischemia Induces Myocardial Dysfunction and Death.

Inhibitors of the ubiquitin-proteasome system improve hemodynamic parameters and decrease the infarct size after ischemia reperfusion. The molecular basis of this protection is not fully understood since most available data report inhibition of the 26 proteasome after ischemia reperfusion. The decrease in cellular ATP levels during ischemia leads to the dissociation of the 26S proteasome into the 19S regulatory complex and the 20S catalytic core, which results in protein degradation independently of ubiquitination. There is scarce information on the activity of the 20S proteasome during cardiac ischemia. Accordingly, the aim of this work was to determine the effects of 30 minutes of ischemia, or 30 min of ischemia followed by 60 minutes of reperfusion on the three main peptidase activities of the 20S proteasome in Langendorff perfused rat hearts. We found that 30 min of ischemia produced a significant increase in the chymotrypsin-like activity of the proteasome, without changes in its caspase-like or trypsin-like activities. In contrast, all three activities were decreased upon reperfusion. Ixazomib, perfused before ischemia at a concentration that reduced the chymotrypsin-like activity to 50% of the control values, without affecting the other proteasomal activities, improved the hemodynamic parameters upon reperfusion and decreased the infarct size. Ixazomib also prevented the 50% reduction in RyR2 content observed after ischemia. The protection was lost, however, when simultaneous inhibition of chymotrypsin-like and caspase-like activities of the proteasome was achieved at higher concentration of ixazomib. Our results suggest that selective inhibition of chymotrypsin-like activity of the proteasome during ischemia preserves key proteins for cardiomyocyte function and exerts a positive impact on cardiac performance after reperfusion.

Protection of the myocardium against ischemia/reperfusion injury by angiotensin-(1–9) through an AT2R and Akt-dependent mechanism

ABSTRACT Angiotensin‐(19), a peptide of the non‐classical renin angiotensin system, has been shown to prevent and revert hypertension and cardiac hypertrophy. We hypothetized that systemic delivery of angiotensin‐(1–9) following myocardial infarction will also be protective and extend to provide protection during reperfusion of the ischemic heart. Adult Sprague Dawley rats were subjected to left anterior descending artery ligation and treated with angiotensin‐(1–9) via osmotic mini‐pump for 2 weeks in the presence or absence of Mas receptor or AT2R antagonists (A779 and PD123319, respectively). Myocardial death and left ventricular function were evaluated after infarction. Infarct size and functional parameters were determined in isolated rat hearts after global ischemia/reperfusion in the presence of angiotensin‐(1–9) plus receptor antagonists or Akt inhibitor at reperfusion. in vitro, neonatal rat ventricular cardiomyocytes underwent simulated ischemia/reperfusion and angiotensin‐(1–9) was co‐incubated with A779, PD123319 or Akt inhibitor. Systemic delivery of angiotensin‐(1–9) significantly decreased cell death and improved left ventricular recovery after in vivo myocardial infarction. Perfusion with the peptide reduced the infarct size and improved functional recovery after ex vivo ischemia/reperfusion. In vitro, angiotensin‐(1–9) decreased cell death in isolated neonatal rat ventricular cardiomyocytes subjected to simulated ischemia/reperfusion. The cardioprotective effects of angiotensin‐(1–9) were blocked by PD123319 and Akti VIII but not by A779. Angiotensin‐(1–9) limits reperfusion‐induced cell death by an AT2R‐ and Aktdependent mechanism. Angiotensin‐(1–9) is a novel strategy to protect against cardiac ischemia/reperfusion injury.