viral-transgene-expression-rodent-hearts-assessment-cardiac

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents ...

University of Ottawa Heart Institute

University of Ottawa

The advent of cellular reprogramming technology has revolutionized biomedical research.  human cardiac myocytes can now be obtained from direct reprogramming of somatic cells (such as fibroblasts), from induced pluripotent stem cells (iPSCs, which are reprogrammed from somatic cells), and from human embryonic stem cells (hESCs). Such  human cardiac myocytes hold great promise for  disease modeling and drug screening and  cell therapy of heart disease. Here, we review the technique advancements for generating  human cardiac myocytes. We also discuss several challenges for the use of such cells in research and regenerative medicine, such as the immature phenotype and heterogeneity of  cardiac myocytes obtained with existing protocols. We focus on the recent advancements in addressing such challenges.

 Human Cardiac Myocytes for Medical Research: Promises and Challenges.

Induced Pluripotent Stem Cell-Based Treatment of Acquired Heart Block: The Battle for Tomorrow Has Begun!

Opioid-induced hyperalgesia (OIH) and allodynia is a well-known phenomenon and refers to the pain sensitization in patients after prolonged opioid exposure. OIH limits the use of opioids in pain control, but the underlying mechanisms are not fully clear. This study investigated the role of mitochondrial Ca uniporter (MCU) in remifentanil (a commonly used opioid analgesic)-induced allodynia. Using a rat model of OIH, we found that incision- and remifentanil-induced mechanical allodynia were remarkably attenuated by pretreatment with Ru360, a specific MCU antagonist, suggesting a critical role of MCU in both incision- and opioid-induced allodynia. In addition, imaging studies with Rhod-2 (a mitochondrial Ca dye) in spinal tissues demonstrated increased mitochondrial Ca level in response to incision and remifentanil infusion, which was attenuated by Ru360. Western blot and immunohistochemistry showed that pNR [phosphorylated N-methyl-D-aspartate (NMDA) receptor] and pERK (phosphorylated extracellular signal-regulated kinase) are increased during both incision-induced hyperalgesia and remifentanil-induced hyperalgesia, and again the increases in pNR and pERK were remarkably attenuated by Ru360. Together, our data demonstrate that MCU plays a critical role in remifentanil-induced postoperative mechanical allodynia, with NMDA receptor and ERK as possible downstream effectors. Our findings provide novel mechanisms for remifentanil-induced mechanical allodynia and encourage future studies to examine the mitochondrial Ca uniporter as a potential therapeutic target for prevention of OIH.

Role of mitochondrial Ca uniporter in remifentanil-induced postoperative allodynia.

Hydrogen sulfide (HS) is endogenously produced in pancreatic ß cells and its level is elevated in diabetes. Here, we report that HS affects insulin secretion via two mechanisms that converge on cytosolic free Ca ([Ca]), a key mediator of insulin exocytosis. Cellular calcium imaging, using Fura-2 or Fluo-4, showed that exposure of INS-1E cells to HS (30-100 μM) reduced both [Ca] levels (by 21.7 ± 2.3%) and oscillation frequency (p < 0.01, n = 4). Consistent with a role of plasma membrane K channels (plasma-K), the effects of HS on [Ca] were blocked by gliclazide (a blocker of plasma-K channels), but were mimicked by diazoxide (an activator of plasma-K channels). Surprisingly, when Ca entry via plasma membrane was inhibited using Ca-free external solutions, HS increased [Ca] by 39.7 ± 3.6% suggesting Ca release from intracellular stores. HS-induced [Ca] increases were abolished by either FCCP (which depletes Ca stored in mitochondria) or cyclosporine A (an inhibitor of mitochondrial permeability transition pore, mPTP) suggesting that HS induces Ca release from mitochondria. Measurement of mitochondrial membrane potential (MMP) suggested that HS causes MMP depolarization, which was blocked by cyclosporine A. Finally, insulin measurements by ELISA indicated that HS decreased insulin release from INS-1E cells, but after plasma membrane Ca entry was blocked by nifedipine, HS-induced mitochondrial Ca release is able to increase insulin release. Together, our results indicate that HS has dual effects on insulin release suggesting that, with different metabolic conditions, HS may differentially modulate the insulin release from pancreatic ß cells and play a role in ß cell dysfunction.

ATP-sensitive K channels and mitochondrial permeability transition pore mediate effects of hydrogen sulfide on cytosolic Ca homeostasis and insulin secretion in β-cells.

Myocardial infarction and heart failure are associated with reduced voltage-gated Na current (I) that promotes arrhythmias and sudden deaths. We have previously shown that the Wnt/β-catenin signalling (Wnt signalling), which is active in heart disease, reduces cardiac I, suggesting that Wnt signalling may be a potential therapeutic target. However, because Wnt signalling is required for the homeostasis of many noncardiac tissues, administration of Wnt inhibitors to heart patients would cause significant side effects. The present study aims to elucidate the molecular mechanisms of cardiac I inhibition by Wnt, which would identify cardiac-specific therapeutic targets.

Direct and Indirect Suppression of Scn5a Gene Expression Mediates Cardiac Na Channel Inhibition by Wnt Signalling.

While encapsulation of cells within protective nanoporous gel cocoons increases cell retention and pro-survival integrin signaling, the influence of cocoon size and intra-capsular cell-cell interactions on therapeutic repair are unknown. Here, we employ a microfluidic platform to dissect the impact of cocoon size and intracapsular cell number on the regenerative potential of transplanted heart explant-derived cells. Deterministic increases in cocoon size boosted the proportion of multicellular aggregates within cocoons, reduced vascular clearance of transplanted cells and enhanced stimulation of endogenous repair. The latter being attributable to cell-cell stimulation of cytokine and extracellular vesicle production while also broadening of the miRNA cargo within extracellular vesicles. Thus, by tuning cocoon size and cell occupancy, the paracrine signature and retention of transplanted cells can be enhanced to promote paracrine stimulation of endogenous tissue repair.

Deterministic paracrine repair of injured myocardium using microfluidic-based cocooning of heart explant-derived cells.

The sinus node (SAN) is the primary pacemaker of the human heart, and abnormalities in its structure or function cause sick sinus syndrome, the most common reason for electronic pacemaker implantation. Here we report that transcription factor GATA6, whose mutations in humans are linked to arrhythmia, is highly expressed in the SAN and its haploinsufficiency in mice results in hypoplastic SANs and rhythm abnormalities. Cell-specific deletion reveals a requirement for GATA6 in various SAN lineages. Mechanistically, GATA6 directly activates key regulators of the SAN genetic program in conduction and nonconduction cells, such as TBX3 and EDN1, respectively. The data identify GATA6 as an important regulator of the SAN and provide a molecular basis for understanding the conduction abnormalities associated with GATA6 mutations in humans. They also suggest that GATA6 may be a potential modifier of the cardiac pacemaker.

GATA6 is a regulator of sinus node development and heart rhythm.

Wnt/β-catenin signaling is activated in the heart after myocardial infarction (MI). This study aims to investigate if β-catenin deletion affects post-MI ion channel gene alterations and ventricular tachycardias (VT). MI was induced by permanent ligation of left anterior descending artery in wild-type (WT) and cardiomyocyte-specific β-catenin knockout (KO) mice. KO mice showed reduced susceptibility to VT (18% vs. 77% in WT) at 8 weeks after MI, associated with reduced scar size and attenuated chamber dilation. qPCR analyses of both myocardial tissues and purified cardiomyocytes demonstrated upregulation of Wnt pathway genes in border and infarct regions after MI, including Wnt ligands (such as Wnt4) and receptors (such as Fzd1 and Fzd2). At 1 week after MI, cardiac sodium channel gene (Scn5a) transcript was reduced in WT but not in KO hearts, consistent with previous studies showing Scn5a inhibition by Wnt/β-catenin signaling. At 8 weeks after MI when Wnt genes have declined, Scn5a returned to near sham levels and K channel gene downregulations were not different between WT and KO mice. This study demonstrated that VT susceptibility in the chronic phase after MI is reduced in mice with cardiomyocyte-specific β-catenin deletion primarily through attenuated structural remodeling, but not ion channel gene alterations.

Aizhu Lu

University of Ottawa Heart Institute

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk