Non-myocytes Research Articles

Abstract Tu008: Efficient Intramyocardial Delivery of EV-Encapsulated AAVs to Target Cardiomyocytes in a Pre-Clinical Swine Model

Background: Adeno-associated viruses (AAV) have been widely used to deliver therapeutic genes to the heart due to their safety and efficacy. However, a major obstacle to their successful gene delivery is the presence of neutralizing antibody (NAb) which form during natural exposure to AAV or following AAV administration. Our recent publication has shown that EV-encapsulated AAVs (EV-AAVs) shields AAVs from NAb neutralization, and overcoming this key roadblock associated with AAV-mediated gene therapy. Hypothesis: In this study, we addressed whether EV-AAVs can target cardiomyocytes in vivo . Aims: To test whether cardiomyocytes effectively uptake EV-AAVs in vivo , in a pre-clinical swine model. Methods and Results: Adult pigs prescreened for a lower NAb titer were randomly assigned to receive intramyocardial administration of either vehicle, AAV6-Luciferase or EV-AAV6-Luciferase. A total of six injections across three injection sites within the left ventricle (LV) were administered per animal. All animals were sacrificed at four weeks and liver and the LV tissues were collected. Luciferase activity assay, flow cytometry analysis of isolated cardiomyocytes (CM) and non-myocytes (NM), and immunofluorescence microscopy were performed to evaluate transgene expression and cellular tropism. Echocardiography was performed at baseline and at 4W to monitor cardiac function. At 4W, EV-AAV6s resulted in a higher luciferase expression in LV compared to nude AAV6 particles. Expression in the LV was higher compared to the liver. Flow cytometry analysis revealed a higher luciferase expression in CM compared to NM, which was corroborated by Immunofluorescence. No differences were detected in the global heart function across groups. Biodistribution of EV-AAV uptake and transgene expression are being studied with intramyocardial and intracoronary delivery mechanisms. Conclusion: EV-AAVs deliver higher amounts of genes than nude AAV6 via intramyocardial administration in a pre-clinical swine model. CMs preferentially uptake EV-AAVs compared to NMs. Our studies suggest that EV-AAVs are a superior cardiac gene delivery vector for future clinical studies.

Nonmyocytes as electrophysiological contributors to cardiac excitation and conduction.

Although cardiac action potential (AP) generation and propagation have traditionally been attributed exclusively to cardiomyocytes (CM), other cell types in the heart are also capable of forming electrically conducting junctions. Interactions between CM and nonmyocytes (NM) enable and modulate each other's activity. This review provides an overview of the current understanding of heterocellular electrical communication in the heart. Although cardiac fibroblasts were initially thought to be electrical insulators, recent studies have demonstrated that they form functional electrical connections with CM in situ. Other NM, such as macrophages, have also been recognized as contributing to cardiac electrophysiology and arrhythmogenesis. Novel experimental tools have enabled the investigation of cell-specific activity patterns in native cardiac tissue, which is expected to yield exciting new insights into the development of novel or improved diagnostic and therapeutic strategies.

Observing and Manipulating Cell-Specific Cardiac Function with Light.

The heart is a complex multicellular organ comprising both cardiomyocytes (CM), which make up the majority of the cardiac volume, and non-myocytes (NM), which represent the majority of cardiac cells. CM drive the pumping action of the heart, triggered via rhythmic electrical activity. NM, on the other hand, have many essential functions including generating extracellular matrix, regulating CM activity, and aiding in repair following injury. NM include neurons and interstitial, immune, and endothelial cells. Understanding the role of specific cell types and their interactions with one another may be key to developing new therapies with minimal side effects to treat cardiac disease. However, assessing cell-type-specific behavior in situ using standard techniques is challenging. Optogenetics enables population-specific observation and control, facilitating studies into the role of specific cell types and subtypes. Optogenetic models targeting the most important cardiac cell types have been generated and used to investigate non-canonical roles of those cell populations, e.g., to better understand how cardiac pacing occurs and to assess potential translational possibilities of optogenetics. So far, cardiac optogenetic studies have primarily focused on validating models and tools in the healthy heart. The field is now in a position where animal models and tools should be utilized to improve our understanding of the complex heterocellular nature of the heart, how this changes in disease, and from there to enable the development of cell-specific therapies and improved treatments.

Optogenetic Modulation of Cardiomyocyte Excitability

Optogenetics is a fast-developing technology, first applied to neuroscience research. A number of studies have begun to transfer the optogenetic approach to cardiovascular research, with a focus on electrical regulation of specific cell types in the heart. There is demand to develop tools to selectively modulate electrical activity in cardiomyocytes (CM) and to interfere with membrane currents in non-myocytes (NM) to advance our understanding of heterocellular-electrotonic coupling in vitro and in vivo. We here report on cell-type specific activation of co-cultured CM and NM using the light-gated cation channel ChR2. Furthermore, we tested three anion-selective channelrhodopsins (ACR) for their potential to inhibit action potential initiation and propagation.Neonatal hearts of the lines WT1-VSFP+;+, αMHC-VSFP+;+ and WT1-ChR2-H134R+;+ were isolated and digested. Cultured cells were transfected with cDNA of ACRs coupled to fluorescent marker proteins.In order to analyse heterocellular coupling we performed whole-cell patch-clamp recordings on CM cocultured with NM from the WT1-ChR2-H134R+;+ line. In a first set of experiments, pulsed ChR2 activation in NM evoked action potentials in patched CM, indicating direct electrical coupling.Next, we tested three different ACR for their potential to hyperpolarise cardiac cells (GtACR1-eGFP, iC++ mCherry, Phobos-mCherry). Transfected cells were patch-clamped in the current-clamp mode to follow light-induced changes in membrane voltage. Notably, ACR activation lead to hyperpolarisation in isolated cardiac NM and to depolarisation in CM.We would like to inhibit cardiac electrical activity by optogenetic hyperpolarisation of NM. As we have shown ACR photocurrents depolarise CM and evoke action potentials, different to the inhibitory effect reported in neurons. We expect that NM, which are connected to CM, have more negative potentials, resulting in depolarising ACR currents also in NM. Therefore, we are looking for a stronger tool to inhibit cardiac activity and plan to generate a light-activated K+-channel.

Cellular Physiology of Rat Cardiac Myocytes in Cardiac Fibrosis: In Vitro Simulation Using the Cardiac Myocyte/Cardiac Non-Myocyte Co-Culture System

An understanding of the cellular physiology of cardiac myocytes (MCs) and non-myocytes (NMCs) may help to explain the mechanisms underlying cardiac hypertrophy. Despite numerous studies using MC/NMC co-culture systems, it is difficult to precisely evaluate the influence of each cell type because of the inherent cellular heterogeneity of such a system. Here we developed a co-culture system using Wistar rat neonatal MCs and NMCs isolated by discontinuous Percoll gradient and adhesion separation methods and cultured on either side of insert well membranes. Co-culture of MCs and NMCs resulted in significant increases in [3H]-leucine incorporation by MCs, in the amount of protein synthesized by MCs, and in the secretion of natriuretic peptides, while the addition of MCs to NMC cultures significantly reduced [3H]-thymidine incorporation by NMCs. Interestingly, the percentage of the brain natriuretic peptide (BNP) component of total natriuretic peptide secreted (atrial natriuretic peptide+BNP) increased as the number of NMCs placed in the MC/NMC co-culture system increased. However, MCs did not affect production of angiotensin II (Ang II) by NMCs or secretion of endothelin-1 and transforming growth factor-beta1 into the MC/NMC co-culture system. This finding was supported by the anti-hypertrophic and anti-fibrotic actions of RNH6270, an active form of olmesartan, on MCs in the MC/NMC co-culture system and on NMCs that may synthesize Ang II in the heart. The present data indicate that cardiac fibrosis may not only facilitate MC hypertrophy (possibly through the local angiotensin system) but may also change particular pathophysiological properties of MCs, such as the secretory pattern of natriuretic peptides.

Effects of urocortin II on neonatal rat cardiac myocytes and non-myocytes

Urocortin (Ucn) II and III, homologous peptides of Ucn that are specific ligands for corticotropin-releasing hormone (CRH) type 2 receptor (CRH-R2), have recently been identified. The present study was designed to elucidate the effects of Ucn II, which is predominantly expressed in rodent heart, on neonatal rat cardiac myocytes (MCs) and cardiac non-myocytes (NMCs). Ucn II increased the incorporation of [ 3H]-leucine into MCs, as well as the accumulation of cAMP and the secretion of atrial natriuretic peptide. However, no significant changes were demonstrated in NMCs or an MC/NMC co-culture system. The effects of Ucn II were attenuated by astressin2-B, a specific antagonist of CRH-R2, and/or H89, an inhibitor of protein kinase A (PKA). These results indicate that Ucn II may be another endogenous cardiovascular substance that acts via CRH-R2 and the cAMP-dependent PKA pathway.

The effects of overexpression of the Na +/Ca 2+ exchanger on calcium regulation in hypertrophied mouse cardiac myocytes

In cardiac hypertrophy and failure it has been shown that the amount of Na/Ca exchanger protein can increase. Several studies have investigated this modification in overt heart failure. However, the role of Na/Ca exchanger overexpression during the development of hypertrophy is unknown. To address this question we investigated Ca 2+ regulation in an early stage of cardiac hypertrophy before signs of heart failure occurred and evaluated the role of Na/Ca exchanger overexpression. Cardiac hypertrophy was induced by a constant infusion of angiotensin II (Ang, 1 μg/min/kg) via an osmotic pump for 14 days. Thereafter, ventricular myocytes from either wild type (NON) or transgenic mice overexpressing the Na/Ca exchanger (TR) were isolated. Myocytes were loaded with indo-1 AM or fluo-4 AM to monitor cytoplasmic [Ca 2+] with all experiments performed at 37 °C. In myocytes exposed to Ang there was an increase in cell capacitance of more than 20% indicating cellular hypertrophy. Ca 2+ transients were prolonged in hypertrophied NON myocytes but not in TR myocytes. Action potentials had a less negative plateau in TR myocytes. Sarcoplasmic reticulum (SR) Ca 2+ content, measured using rapid caffeine application, was greater in TR myocytes but unaffected by hypertrophy. Ca 2+ spark frequency was significantly greater in TR. Na/Ca exchanger overexpression prevented the prolongation of the Ca 2+ transient observed in hypertrophy and maintained a similar SR Ca 2+ leak suggesting a compensatory role in Ca 2+ regulation in hypertrophied cardiac myocytes from transgenic mice. We suggest this compensatory effect is mediated by increased SR Ca 2+ content and faster Ca 2+ removal via the Na/Ca exchanger.

Urocortin has cell-proliferative effects on cardiac non-myocytes

Urocortin (Ucn) is a member of the corticotropin-releasing hormone (CRH)-related peptides that has been reported to have cardiac inotropic and hypertrophic effects. In addition, Ucn mRNA was expressed in cardiac myocytes (MCs) and Ucn was suggested to have cardioprotective effects. Recently, it was reported that Ucn mRNA was expressed in cardiac non-myocytes (NMCs). Based on these facts, Ucn is assumed to affect not only MCs but also NMCs in an autocrine fashion. The present study was designed to elucidate a pathophysiological role of Ucn on NMCs. NMCs were prepared by the discontinuous Percoll gradient and adhesion method. Ucn increased [ 3H]-thymidine uptake into NMCs. Ucn also enhanced endothelin-1-induced increase of [ 3H]-thymidine uptake into NMCs. Effects of Ucn on [ 3H]-thymidine uptake into NMCs were significantly abolished by the protein kinase A inhibitor, H89 (10 −5 M), but not by a competitive antagonist of CRH receptors, astressin (10 −5 M). Ucn also increased intracellular cAMP accumulation more potently than CRH on a molar basis. Finally, both MCs and NMCs also secreted Ucn. Together with the recent findings, at least in NMCs, these data suggest that Ucn could exert its own actions via the cAMP signaling pathway, but not through known CRH type 2 receptors, in an autocrine fashion. In conclusion, the present study indicated that Ucn was secreted not only from MCs but also from NMCs and that the primary source of Ucn acting on heart was the heart itself. On the other hand, Ucn could proliferate NMCs as well as MCs, suggesting that Ucn could be involved in cardiac hypertrophy and fibrosis, i.e., cardiac remodeling, in spite of its putative cardioprotective actions.

Overexpression of the Na(+)/Ca(2+) exchanger and inhibition of the sarcoplasmic reticulum Ca(2+)-ATPase in ventricular myocytes from transgenic mice.

Myocytes from failing hearts produce slower and smaller Ca(2+) transients associated with reduction in expression of sarcoplasmic reticulum (SR) Ca(2+) ATPase and an overexpression of Na(+)/Ca(2+) exchanger. Since the physiological role of both these proteins is competing for, and removing, Ca(2+) from the cytoplasm, overexpression of the exchanger may compensate for less effective SR Ca(2+) uptake. This study demonstrates this compensatory effect and provides a quantitative description of the results. Ventricular myocytes from transgenic mice overexpressing the Na(+)/Ca(2+) exchanger (TR) and nontransgenic littermates (NON) were used. Cell shortening, cytoplasmic [Ca] (using indo-1 AM) and electrophysiological parameters were monitored. TR myocytes displayed faster Ca(2+) transients and twitches compared with NON myocytes. Superfusion with thapsigargin prolonged the time-course of Ca(2+) transients of TR myocytes until these were equal to the ones measured in NON myocytes. The amount of SR Ca(2+)-ATPase (SERCA) inhibition needed to obtain such transients was calculated as a function of V(max) for the Ca(2+) flux via SERCA and found to be 28%. In TR myocytes V(max) for the Ca(2+) flux via Na(+)/Ca(2+)exchange was 240% of NON myocytes. When Ca(2+) transients in TR myocytes were slowed by thapsigargin to similar values to the ones recorded in NON myocytes, SR Ca(2+) content was also correspondingly reduced. The results suggest that in pathophysiological conditions where there is a reduction in SERCA function, overexpression of Na(+)/Ca(2+) exchanger can compensate and allow normal Ca(2+) homeostasis to be maintained. In mouse ventricular myocytes a 2.4-fold increase in Na(+)/Ca(2+) exchange activity compensates for a reduction in SERCA function by 28% so maintaining the duration of the Ca(2+) transient.

Urocortin, a member of the corticotropin-releasing factor family, in normal and diseased heart.

In the present study we investigated the form of expression, action, second messenger, and the cellular location of urocortin, a member of the corticotropin-releasing factor (CRF) family, in the heart. Urocortin mRNA, as shown by quantitative RT-PCR analysis, is expressed in the cultured rat cardiac nonmyocytes (NMC) as well as myocytes (MC) in the heart, whereas CRF receptor type 2beta (CRF-R2beta), presumed urocortin receptor mRNA, is predominantly expressed in MC compared with NMC. Urocortin mRNA expression is higher in left ventricular (LV) hypertrophy than in normal LV, whereas CRF-R2beta mRNA expression is markedly depressed in LV hypertrophy compared with normal LV. Urocortin more potently increased the cAMP levels in both MC and NMC than did CRF, and its effect was more potent in MC than in NMC. Urocortin significantly increased protein synthesis by [(14)C]Phe incorporations and atrial natriuretic peptide secretion in MC and collagen and increased DNA synthesis by [(3)H]prolin and [(3)H]Thy incorporations in NMC. An immunohistochemical study revealed that urocortin immunoreactivity was observed in MC in the normal human heart and that it was more intense in the MC of the human failing heart than in MC of the normal heart. These results, together with the recent evidence of urocortin for positive inotropic action, suggest that increased urocortin in the diseased heart may modulate the pathophysiology of cardiac hypertrophy or failing heart, at least in part, via cAMP signaling pathway.

The effects of sarpogrelate on cardiomyocyte hypertrophy

Sarpogrelate was developed as an antiplatelet agent antagonizing 5-hydroxytryptamine (5-HT) receptors. It had been reported that 5-HT receptors were expressed in cardiovascular system, and that sarpogrelate had antihypertrophic effects in vascular smooth muscle cells. Cardiac hypertrophy is a major problem in cardiac diseases, so the present study was designed to elucidate the effects of sarpogrelate on cardiac hypertrophy. Cultured rat cardiomyocytes (MCs) and cardiac nonmyocytes (NMCs) were prepared by Percoll gradient and adhesion method and MCs were incubated with (MCs/NMCs) or without NMCs. As an index of protein synthesis of MCs, [ 3H]-leucine uptake into MCs and MCs/NMCs was measured. Sarpogrelate decreased [ 3H]-leucine uptake into MCs (maximun 62.6±20.6% of control at 10 −4M, p<0.05 vs. control). Sarpogrelate also significantly attenuated angiotensin-II- and endothelin-1-induced [ 3H]-leucine uptake. These results indicated that sarpogrelate might have antihypertrophic effects and could be a useful aid for cardiovascular disease.

Interaction of myocytes and nonmyocytes is necessary for mechanical stretch to induce ANP/BNP production in cardiocyte culture.

In cardiac hypertrophy or ventricular remodeling, enlargement of myocytes and interstitial or perivascular fibrosis are observed simultaneously, which suggests an interaction between cardiac myocytes and fibroblasts. In this study we examined the mechanism of cyclic mechanical stretch-induced myocytic hypertrophy, focusing on the interaction between myocytes and cardiac nonmyocytes, mostly fibroblasts. Ventricular myocytes (MCs) and cardiac nonmyocytes (NMCs) were separately extracted from neonatal rat ventricles by the discontinuous Percoll gradient method and primary cultures of cardiac cells were prepared. When MCs were co-cultured with NMCs, the size of MCs and the ANP/BNP secretion were significantly increased. This hypertrophic change of MCs in the co-culture was significantly suppressed by BQ-123, an endothelin-A (ETA) receptor antagonist. Cyclic stretch did not induce hypertrophic responses in MC culture. However, it further increased ANP/BNP production in MC-NMC co-culture (2.2-fold and 2.1-fold increases vs. non-stretch group after 48-h incubation). This increase in ANP/BNP production in the co-culture was significantly suppressed by CV-11974, an angiotensin II (Ang II) type 1 receptor antagonist. This study raises the possibility that NMCs regulate cardiocyte hypertrophy via secretion of endothelin-1 and that Ang II is involved in the interaction between MCs and NMCs during the course of hypertrophic response of cardiocytes to mechanical stretch.

Significance of ventricular myocytes and nonmyocytes interaction during cardiocyte hypertrophy: evidence for endothelin-1 as a paracrine hypertrophic factor from cardiac nonmyocytes.

In cardiac hypertrophy, both excessive enlargement of cardiac myocytes and progressive interstitial fibrosis are well known to occur simultaneously. In the present study, to investigate the interaction between ventricular myocytes (MCs) and cardiac nonmyocytes (NMCs), mostly fibroblasts, during cardiocytes hypertrophy, we examined the change in cell size and gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in cultured MCs as markers for hypertrophy in the neonatal rat ventricular cardiac cell culture system. The size of cultured MCs significantly increased in the MC-NMC coculture. Concomitantly, secretions of ANP and BNP into culture media were significantly increased in the MC-NMC coculture compared with in the MC culture (with the possible contamination of NMC <1% of MC). Moreover, in the MC culture, enlargement of MC and an increase in ANP and BNP secretions were induced by treatment with conditioned media of the NMC culture. A considerable amount of endothelin (ET)-1 production was detected in the NMC-conditioned media. BQ-123, an ET-A receptor antagonist, and bosentan, a nonselective ET receptor antagonist, significantly blocked the hypertrophic response of MCs induced by treatment with NMC-conditioned media. Angiotensin II (Ang II) (10(-10) to 10(-6) mol/L) and transforming growth factor-beta1 (TGF-beta1) (10(-13) to 10(-9) mol/L), both of which are known to be cardiac hypertrophic factors, did not induce hypertrophy in MC culture, but both Ang II and TGF-beta1 increased the size of MCs and augmented ANP and BNP productions in the MC-NMC coculture. This hypertrophic activity of Ang II and TGF-beta1 was associated with the potentiation of ET-1 production in the MC-NMC coculture, and the effect of Ang II or TGF-beta1 on the secretions of ANP and BNP in the coculture was significantly suppressed by pretreatment with BQ-123. These results demonstrate that NMCs regulate MC hypertrophy at least partially via ET-1 secretion and that the interaction between MCs and NMCs plays a critical role during the process of Ang II- or TGF-beta1-induced cardiocyte hypertrophy.