Myeloperoxidase As a Multifaceted Target for Cardiovascular Protection.

Significance: Myeloperoxidase (MPO) is a heme peroxidase that is primarily expressed by neutrophils. It has the capacity to generate several reactive species, essential for its inherent antimicrobial activity and innate host defense. Dysregulated MPO release, however, can lead to tissue damage, as seen in several diseases. Increased MPO levels in circulation are therefore widely associated with conditions of increased oxidative stress and inflammation. Recent Advances: Several studies have shown a strong correlation between MPO and cardiovascular disease (CVD), through which elevated levels of circulating MPO are linked to poor prognosis with increased risk of CVD-related mortality. Accordingly, circulating MPO is considered a "high-risk" biomarker for patients with acute coronary syndrome, atherosclerosis, heart failure, hypertension, and stroke, thereby implicating MPO as a multifaceted target for cardiovascular protection. Consistently, recent studies that target MPO in animal models of CVD have demonstrated favorable outcomes with regard to disease progression. Critical Issues: Although most of these studies have established a critical link between circulating MPO and worsening cardiac outcomes, the mechanisms by which MPO exerts its detrimental effects in CVD remain unclear. Future Directions: Elucidating the mechanisms by which elevated MPO leads to poor prognosis and, conversely, investigating the beneficial effects of therapeutic MPO inhibition on alleviating disease phenotype will facilitate future MPO-targeted clinical trials for improving CVD-related outcomes.

Targeting Mitochondrial Fission Using Mdivi-1 in A Clinically Relevant Large Animal Model of Acute Myocardial Infarction: A Pilot Study.

Background: New treatments are needed to reduce myocardial infarct size (MI) and prevent heart failure (HF) following acute myocardial infarction (AMI), which are the leading causes of death and disability worldwide. Studies in rodent AMI models showed that genetic and pharmacological inhibition of mitochondrial fission, induced by acute ischemia and reperfusion, reduced MI size. Whether targeting mitochondrial fission at the onset of reperfusion is also cardioprotective in a clinically-relevant large animal AMI model remains to be determined. Methods: Adult pigs (30-40 kg) were subjected to closed-chest 90-min left anterior descending artery ischemia followed by 72 h of reperfusion and were randomized to receive an intracoronary bolus of either mdivi-1 (1.2 mg/kg, a small molecule inhibitor of the mitochondrial fission protein, Drp1) or vehicle control, 10-min prior to reperfusion. The left ventricular (LV) size and function were both assessed by transthoracic echocardiography prior to AMI and after 72 h of reperfusion. MI size and the area-at-risk (AAR) were determined using dual staining with Tetrazolium and Evans blue. Heart samples were collected for histological determination of fibrosis and for electron microscopic analysis of mitochondrial morphology. Results: A total of 14 pigs underwent the treatment protocols (eight control and six mdivi-1). Administration of mdivi-1 immediately prior to the onset of reperfusion did not reduce MI size (MI size as % of AAR: Control 49.2 ± 8.6 vs. mdivi-1 50.5 ± 11.4; p = 0.815) or preserve LV systolic function (LV ejection fraction %: Control 67.5 ± 0.4 vs. mdivi-1 59.6 ± 0.6; p = 0.420), when compared to vehicle control. Similarly, there were no differences in mitochondrial morphology or myocardial fibrosis between mdivi-1 and vehicle control groups. Conclusion: Our pilot study has shown that treatment with mdivi-1 (1.2 mg/kg) at the onset of reperfusion did not reduce MI size or preserve LV function in the clinically-relevant closed-chest pig AMI model. A larger study, testing different doses of mdivi-1 or using a more specific Drp1 inhibitor are required to confirm these findings.

Fatty acid metabolism driven mitochondrial bioenergetics promotes advanced developmental phenotypes in human induced pluripotent stem cell derived cardiomyocytes.

BACKGROUND: Preferential utilization of fatty acids for ATP production represents an advanced metabolic phenotype in developing cardiomyocytes. We investigated whether this phenotype could be attained in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) and assessed its influence on mitochondrial morphology, bioenergetics, respiratory capacity and ultra-structural architecture. METHODS AND RESULTS: Whole-cell proteome analysis of day 14 and day 30-CMs maintained in glucose media revealed a positive influence of extended culture on mitochondria-related processes that primed the day 30-CMs for fatty acid metabolism. Supplementing the day 30-CMs with palmitate/oleate (fatty acids) significantly enhanced mitochondrial remodeling, oxygen consumption rates and ATP production. Metabolomic analysis upon fatty acid supplementation revealed a ß-oxidation fueled ATP elevation that coincided with presence of junctional complexes, intercalated discs, t-tubule-like structures and adult isoform of cardiac troponin T. In contrast, glucose-maintained day 30-CMs continued to harbor underdeveloped ultra-structural architecture and more subdued bioenergetics, constrained by suboptimal mitochondria development. CONCLUSION: The advanced metabolic phenotype of preferential fatty acid utilization was attained in hiPSC-CMs, whereby fatty acid driven ß-oxidation sustained cardiac bioenergetics and respiratory capacity resulting in ultra-structural and functional characteristics similar to those of developmentally advanced cardiomyocytes. Better understanding of mitochondrial bioenergetics and ultra-structural adaptation associated with fatty acid metabolism has important implications in the study of cardiac physiology that are associated with late-onset mitochondrial and metabolic adaptations.

ErbB Receptor Tyrosine Kinase: A Molecular Switch Between Cardiac and Neuroectoderm Specification in Human Pluripotent Stem Cells.

Mechanisms determining intrinsic differentiation bias inherent to human pluripotent stem cells (hPSCs) toward cardiogenic fate remain elusive. We evaluated the interplay between ErbB4 and Epidemal growth factor receptor (EGFR or ErbB1) in determining cardiac differentiation in vitro as these receptor tyrosine kinases are key to heart and brain development in vivo. Our results demonstrate that during cardiac differentiation, cell fate biases exist in hPSCs due to cardiac/neuroectoderm divergence post cardiac mesoderm stage. Stage-specific up-regulation of EGFR in concert with persistent Wnt3a signaling post cardiac mesoderm favors commitment toward neural progenitor cells (NPCs). Inhibition of EGFR abrogates these effects with enhanced (>twofold) cardiac differentiation efficiencies by increasing proliferation of Nkx2-5 expressing cardiac progenitors while reducing proliferation of Sox2 expressing NPCs. Forced overexpression of ErbB4 rescued cardiac commitment by augmenting Wnt11 signaling. Convergence between EGFR/ErbB4 and canonical/noncanonical Wnt signaling determines cardiogenic fate in hPSCs. Stem Cells 2016;34:2461-2470.

Electrotonic coupled metabolic purification of chick cardiomyocytes.

Cardiomyocytes isolated from chick and rodent are widely used in studying cardiac physiology. However, contaminating non-cardiomyocytes are an inherent problem that hinders downstream analysis. Here, we report a novel electrical stimulation coupled with metabolic selection method using cytosine arabinoside (AraC) to efficiently eliminate contaminating cells in isolating chick embryonic cardiomyocytes. Compared with conventional methods of pre-plating or AraC alone, electrical stimulation coupled with AraC increased the percentage purity of cardiomyocytes by 2-6-fold with added effect of improved contractile function and maturation. This simple method could be useful in isolating and maintaining purified cardiomyocytes for long-term studies of cardiac physiology.