Determinants of electrical propagation and propagation block in Arrhythmogenic Cardiomyopathy.

Gap junction and ion channel remodeling occur early in Arrhythmogenic Cardiomyopathy (ACM), but their pathogenic consequences have not been elucidated. Here, we identified the arrhythmogenic substrate, consisting of propagation slowing and conduction block, in ACM models expressing two different desmosomal gene variants. Neonatal rat ventricular myocytes were transduced to express variants in genes encoding desmosomal proteins plakoglobin or plakophilin-2. Studies were performed in engineered cells and anisotropic tissues to quantify changes in conduction velocity, formation of unidirectional propagation, cell-cell electrical coupling, and ion currents. Conduction velocity decreased by 71% and 63% in the two ACM models. SB216763, an inhibitor of glycogen synthase kinase-3 beta, restored conduction velocity to near normal levels. Compared to control, both ACM models showed greater propensity for unidirectional conduction block, which increased further at greater stimulation frequencies. Cell-cell electrical conductance measured in cell pairs was reduced by 86% and 87% in the two ACM models. Computer modeling showed close correspondence between simulated and experimentally determined changes in conduction velocity. The simulation identified that reduced cell-cell electrical coupling was the dominant factor leading to slow conduction, while the combination of reduced cell-cell electrical coupling, reduced sodium current and inward rectifier potassium current explained the development of unidirectional block. Expression of two different ACM variants markedly reduced cell-cell electrical coupling and conduction velocity, and greatly increased the likelihood of developing unidirectional block - both key features of arrhythmogenesis. This study provides the first quantitative analysis of cellular electrophysiological changes leading to the substrate of reentrant arrhythmias in early stage ACM.

Cardiac T-Tubule Microanatomy and Function.

Unique to striated muscle cells, transverse tubules (t-tubules) are membrane organelles that consist of sarcolemma penetrating into the myocyte interior, forming a highly branched and interconnected network. Mature t-tubule networks are found in mammalian ventricular cardiomyocytes, with the transverse components of t-tubules occurring near sarcomeric z-discs. Cardiac t-tubules contain membrane microdomains enriched with ion channels and signaling molecules. The microdomains serve as key signaling hubs in regulation of cardiomyocyte function. Dyad microdomains formed at the junctional contact between t-tubule membrane and neighboring sarcoplasmic reticulum are critical in calcium signaling and excitation-contraction coupling necessary for beat-to-beat heart contraction. In this review, we provide an overview of the current knowledge in gross morphology and structure, membrane and protein composition, and function of the cardiac t-tubule network. We also review in detail current knowledge on the formation of functional membrane subdomains within t-tubules, with a particular focus on the cardiac dyad microdomain. Lastly, we discuss the dynamic nature of t-tubules including membrane turnover, trafficking of transmembrane proteins, and the life cycles of membrane subdomains such as the cardiac BIN1-microdomain, as well as t-tubule remodeling and alteration in diseased hearts. Understanding cardiac t-tubule biology in normal and failing hearts is providing novel diagnostic and therapeutic opportunities to better treat patients with failing hearts.

A canine model of chronic ischemic heart failure.

Preclinical large animal models of chronic heart failure (HF) are crucial to both understanding pathological remodeling and translating fundamental discoveries into novel therapeutics for HF. Canine models of ischemic cardiomyopathy are historically limited by either high early mortality or failure to develop chronic heart failure. Twenty-nine healthy adult dogs (30 ± 4 kg, 15/29 male) underwent thoracotomy followed by one of three types of left anterior descending (LAD) coronary artery ligation procedures: group 1 (n = 4) (simple LAD: proximal and distal LAD ligation); group 2 (n = 14) (simple LAD plus lateral wall including ligation of the distal first diagonal and proximal first obtuse marginal); and group 3 (n = 11) (total LAD devascularization or TLD: simple LAD plus ligation of proximal LAD branches to both the right and left ventricles). Dogs were followed until chronic severe HF developed defined as left ventricular ejection fraction (LVEF) < 40% and NH2-terminal-prohormone B-type natriuretic peptide (NT-proBNP) > 900 pmol/L. Overall early survival (48-h postligation) in 29 dogs was 83% and the survival rate at postligation 5 wk was 69%. Groups 1 and 2 had 100% and 71% early survival, respectively, yet only a 50% success rate of developing chronic HF. Group 3 had excellent survival at postligation 48 h (91%) and a 100% success in the development of chronic ischemic HF. The TLD approach, which limits full LAD and collateral flow to its perfusion bed, provides excellent early survival and reliable development of chronic ischemic HF in canine hearts.NEW & NOTEWORTHY The novel total left anterior descending devascularization (TLD) approach in a canine ischemic heart failure model limits collateral flow in the ischemic zone and provides excellent early survival and repeatable development of chronic ischemic heart failure in the canine heart. This work provides a consistent large animal model for investigating heart failure mechanisms and testing novel therapeutics.

The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles.

Microparticles (MPs) are cell-cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT pathway for MP biogenesis in mammalian cardiac ventricular cells, identifying elements of a pathway by which cytoplasmic cBIN1 is released into blood.

GJA1-20k Arranges Actin to Guide Cx43 Delivery to Cardiac Intercalated Discs.

RATIONALE: Delivery of Cx43 (connexin 43) to the intercalated disc is a continuous and rapid process critical for intercellular coupling. By a pathway of targeted delivery involving microtubule highways, vesicles of Cx43 hemichannels are efficiently trafficked to adherens junctions at intercalated discs. It has also been identified that actin provides rest stops for Cx43 forward trafficking and that Cx43 has a 20 kDa internally translated small C terminus isoform, GJA1-20k (Gap Junction Protein Alpha 1- 20 kDa), which is required for full-length Cx43 trafficking, but by an unknown mechanism. OBJECTIVE: We explored the mechanism by which the GJA1-20k isoform is required for full-length Cx43 forward trafficking to intercalated discs. METHODS AND RESULTS: Using an in vivo Adeno-associated virus serotype 9-mediated gene transfer system, we confirmed in whole animal that GJA1-20k markedly increases endogenous myocardial Cx43 gap junction plaque size at the intercalated discs. In micropatterned cell pairing systems, we found that exogenous GJA1-20k expression stabilizes filamentous actin without affecting actin protein expression and that GJA1-20k complexes with both actin and tubulin. We also found that filamentous actin regulates microtubule organization as inhibition of actin polymerization with a low dose of latrunculin A disrupts the targeting of microtubules to cell-cell junctions. GJA1-20k protects actin filament from latrunculin A disruption, preserving microtubule trajectory to the cell-cell border. For therapeutic implications, we found that prior in vivo Adeno-associated virus serotype 9-mediated gene delivery of GJA1-20k to the heart protects Cx43 localization to the intercalated discs against acute ischemic injury. CONCLUSIONS: The internally translated GJA1-20k isoform stabilizes actin filaments, which guides growth trajectories of the Cx43 microtubule trafficking machinery, increasing delivery of Cx43 hemichannels to cardiac intercalated discs. Exogenous GJA1-20k helps to maintain cell-cell coupling in instances of anticipated myocardial ischemia.

Intracellular trafficking pathways of Cx43 gap junction channels.

Gap Junction (GJ) channels, including the most common Connexin 43 (Cx43), have fundamental roles in excitable tissues by facilitating rapid transmission of action potentials between adjacent cells. For instance, synchronization during each heartbeat is regulated by these ion channels at the cardiomyocyte cell-cell border. Cx43 protein has a short half-life, and rapid synthesis and timely delivery of those proteins to particular subdomains are crucial for the cellular organization of gap junctions and maintenance of intracellular coupling. Impairment in gap junction trafficking contributes to dangerous complications in diseased hearts such as the arrhythmias of sudden cardiac death. Of recent interest are the protein-protein interactions with the Cx43 carboxy-terminus. These interactions have significant impact on the full length Cx43 lifecycle and also contribute to trafficking of Cx43 as well as possibly other functions. We are learning that many of the known non-canonical roles of Cx43 can be attributed to the recently identified six endogenous Cx43 truncated isoforms which are produced by internal translation. In general, alternative translation is a new leading edge for proteome expansion and therapeutic drug development. This review highlights recent mechanisms identified in the trafficking of gap junction channels, involvement of other proteins contributing to the delivery of channels to the cell-cell border, and understanding of possible roles of the newly discovered alternatively translated isoforms in Cx43 biology. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

A 14-3-3 mode-1 binding motif initiates gap junction internalization during acute cardiac ischemia.

Altered phosphorylation and trafficking of connexin 43 (Cx43) during acute ischemia contributes to arrhythmogenic gap junction remodeling, yet the critical sequence and accessory proteins necessary for Cx43 internalization remain unresolved. 14-3-3 proteins can regulate protein trafficking, and a 14-3-3 mode-1 binding motif is activated upon phosphorylation of Ser373 of the Cx43 C-terminus. We hypothesized that Cx43(Ser373) phosphorylation is important to pathological gap junction remodeling. Immunofluorescence in human heart reveals the enrichment of 14-3-3 proteins at intercalated discs, suggesting interaction with gap junctions. Knockdown of 14-3-3τ in cell lines increases gap junction plaque size at cell-cell borders. Cx43(S373A) mutation prevents Cx43/14-3-3 complexing and stabilizes Cx43 at the cell surface, indicating avoidance of degradation. Using Langendorff-perfused mouse hearts, we detect phosphorylation of newly internalized Cx43 at Ser373 and Ser368 within 30 min of no-flow ischemia. Phosphorylation of Cx43 at Ser368 by protein kinase C and Ser255 by mitogen-activated protein kinase has previously been implicated in Cx43 internalization. The Cx43(S373A) mutant is resistant to phosphorylation at both these residues and does not undergo ubiquitination, revealing Ser373 phosphorylation as an upstream gatekeeper of a posttranslational modification cascade necessary for Cx43 internalization. Cx43(Ser373) phosphorylation is a potent target for therapeutic interventions to preserve gap junction coupling in the stressed myocardium.

3-OST-7 regulates BMP-dependent cardiac contraction.

The 3-O-sulfotransferase (3-OST) family catalyzes rare modifications of glycosaminoglycan chains on heparan sulfate proteoglycans, yet their biological functions are largely unknown. Knockdown of 3-OST-7 in zebrafish uncouples cardiac ventricular contraction from normal calcium cycling and electrophysiology by reducing tropomyosin4 (tpm4) expression. Normal 3-OST-7 activity prevents the expansion of BMP signaling into ventricular myocytes, and ectopic activation of BMP mimics the ventricular noncontraction phenotype seen in 3-OST-7 depleted embryos. In 3-OST-7 morphants, ventricular contraction can be rescued by overexpression of tropomyosin tpm4 but not by troponin tnnt2, indicating that tpm4 serves as a lynchpin for ventricular sarcomere organization downstream of 3-OST-7. Contraction can be rescued by expression of 3-OST-7 in endocardium, or by genetic loss of bmp4. Strikingly, BMP misregulation seen in 3-OST-7 morphants also occurs in multiple cardiac noncontraction models, including potassium voltage-gated channel gene, kcnh2, affected in Romano-Ward syndrome and long-QT syndrome, and cardiac troponin T gene, tnnt2, affected in human cardiomyopathies. Together these results reveal 3-OST-7 as a key component of a novel pathway that constrains BMP signaling from ventricular myocytes, coordinates sarcomere assembly, and promotes cardiac contractile function.

Role for myosin-V motor proteins in the selective delivery of Kv channel isoforms to the membrane surface of cardiac myocytes.

RATIONALE: Kv1.5 (KCNA5) mediates the ultra-rapid delayed rectifier current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation, Kv1.5 has emerged as a promising target for the treatment of atrial fibrillation. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known. OBJECTIVE: To investigate the role of the unconventional myosin-V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5. METHODS AND RESULTS: Western blot analysis showed MYO5A and MYO5B expression in the heart, whereas disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and short hairpin RNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and connexin-43 but not potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, whereas MYO5B regulated postendocytic recycling. Overexpression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling. CONCLUSIONS: MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current and, consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias.

Na+ channel function, regulation, structure, trafficking and sequestration.

This paper is the second of a series of three reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on Na(+) channel function and regulation, Na(+) channel structure and function, and Na(+) channel trafficking, sequestration and complexing.

Multilayered regulation of cardiac ion channels.

Essential to beat-to-beat heart function is the ability for cardiomyocytes to propagate electrical excitation and generate contractile force. Both excitation and contractility depend on specific ventricular ion channels, which include the L-type calcium channel (LTCC) and the connexin 43 (Cx43) gap junction. Each of these two channels is localized to a distinct subdomain of the cardiomyocyte plasma membrane. In this review, we focus on regulatory mechanisms that govern the lifecycles of LTCC and Cx43, from their biogenesis in the nucleus to directed delivery to T-tubules and intercalated discs, respectively. We discuss recent findings on how alternative promoter usage, tissue-specific transcription, and alternative splicing determine precise ion channel expression levels within a cardiomyocyte. Moreover, recent work on microtubule and actin-dependent trafficking for Cx43 and LTCC are introduced. Lastly, we discuss how human cardiac disease phenotypes can be attributed to defects in distinct mechanisms of channel regulation at the level of gene expression and channel trafficking. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Actin cytoskeleton rest stops regulate anterograde traffic of connexin 43 vesicles to the plasma membrane.

RATIONALE: The intracellular trafficking of connexin 43 (Cx43) hemichannels presents opportunities to regulate cardiomyocyte gap junction coupling. Although it is known that Cx43 hemichannels are transported along microtubules to the plasma membrane, the role of actin in Cx43 forward trafficking is unknown. OBJECTIVE: We explored whether the actin cytoskeleton is involved in Cx43 forward trafficking. METHODS AND RESULTS: High-resolution imaging reveals that Cx43 vesicles colocalize with nonsarcomeric actin in adult cardiomyocytes. Live-cell fluorescence imaging reveals Cx43 vesicles as stationary or traveling slowly (average speed 0.09 µm/s) when associated with actin. At any time, the majority (81.7%) of vesicles travel at subkinesin rates, suggesting that actin is important for Cx43 transport. Using Cx43 containing a hemagglutinin tag in the second extracellular loop, we developed an assay to detect transport of de novo Cx43 hemichannels to the plasma membrane after release from Brefeldin A-induced endoplasmic reticulum/Golgi vesicular transport block. Latrunculin A (for specific interference of actin) was used as an intervention after reinitiation of vesicular transport. Disruption of actin inhibits delivery of Cx43 to the cell surface. Moreover, using the assay in primary cardiomyocytes, actin inhibition causes an 82% decrease (P<0.01) in de novo endogenous Cx43 delivery to cell-cell borders. In Langendorff-perfused mouse heart preparations, Cx43/ß-actin complexing is disrupted during acute ischemia, and inhibition of actin polymerization is sufficient to reduce levels of Cx43 gap junctions at intercalated discs. CONCLUSIONS: Actin is a necessary component of the cytoskeleton-based forward trafficking apparatus for Cx43. In cardiomyocytes, Cx43 vesicles spend a majority of their time pausing at nonsarcomeric actin rest stops when not undergoing microtubule-based transport to the plasma membrane. Deleterious effects on this interaction between Cx43 and the actin cytoskeleton during acute ischemia contribute to losses in Cx43 localization at intercalated discs.

Iroquois homeobox gene 3 establishes fast conduction in the cardiac His-Purkinje network.

Rapid electrical conduction in the His-Purkinje system tightly controls spatiotemporal activation of the ventricles. Although recent work has shed much light on the regulation of early specification and morphogenesis of the His-Purkinje system, less is known about how transcriptional regulation establishes impulse conduction properties of the constituent cells. Here we show that Iroquois homeobox gene 3 (Irx3) is critical for efficient conduction in this specialized tissue by antithetically regulating two gap junction-forming connexins (Cxs). Loss of Irx3 resulted in disruption of the rapid coordinated spread of ventricular excitation, reduced levels of Cx40, and ectopic Cx43 expression in the proximal bundle branches. Irx3 directly represses Cx43 transcription and indirectly activates Cx40 transcription. Our results reveal a critical role for Irx3 in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart.

BIN1 localizes the L-type calcium channel to cardiac T-tubules.

The BAR domain protein superfamily is involved in membrane invagination and endocytosis, but its role in organizing membrane proteins has not been explored. In particular, the membrane scaffolding protein BIN1 functions to initiate T-tubule genesis in skeletal muscle cells. Constitutive knockdown of BIN1 in mice is perinatal lethal, which is associated with an induced dilated hypertrophic cardiomyopathy. However, the functional role of BIN1 in cardiomyocytes is not known. An important function of cardiac T-tubules is to allow L-type calcium channels (Cav1.2) to be in close proximity to sarcoplasmic reticulum-based ryanodine receptors to initiate the intracellular calcium transient. Efficient excitation-contraction (EC) coupling and normal cardiac contractility depend upon Cav1.2 localization to T-tubules. We hypothesized that BIN1 not only exists at cardiac T-tubules, but it also localizes Cav1.2 to these membrane structures. We report that BIN1 localizes to cardiac T-tubules and clusters there with Cav1.2. Studies involve freshly acquired human and mouse adult cardiomyocytes using complementary immunocytochemistry, electron microscopy with dual immunogold labeling, and co-immunoprecipitation. Furthermore, we use surface biotinylation and live cell confocal and total internal fluorescence microscopy imaging in cardiomyocytes and cell lines to explore delivery of Cav1.2 to BIN1 structures. We find visually and quantitatively that dynamic microtubules are tethered to membrane scaffolded by BIN1, allowing targeted delivery of Cav1.2 from the microtubules to the associated membrane. Since Cav1.2 delivery to BIN1 occurs in reductionist non-myocyte cell lines, we find that other myocyte-specific structures are not essential and there is an intrinsic relationship between microtubule-based Cav1.2 delivery and its BIN1 scaffold. In differentiated mouse cardiomyocytes, knockdown of BIN1 reduces surface Cav1.2 and delays development of the calcium transient, indicating that Cav1.2 targeting to BIN1 is functionally important to cardiac calcium signaling. We have identified that membrane-associated BIN1 not only induces membrane curvature but can direct specific antegrade delivery of microtubule-transported membrane proteins. Furthermore, this paradigm provides a microtubule and BIN1-dependent mechanism of Cav1.2 delivery to T-tubules. This novel Cav1.2 trafficking pathway should serve as an important regulatory aspect of EC coupling, affecting cardiac contractility in mammalian hearts.

Cardiac conduction is required to preserve cardiac chamber morphology.

Electrical cardiac forces have been previously hypothesized to play a significant role in cardiac morphogenesis and remodeling. In response to electrical forces, cultured cardiomyocytes rearrange their cytoskeletal structure and modify their gene expression profile. To translate such in vitro data to the intact heart, we used a collection of zebrafish cardiac mutants and transgenics to investigate whether cardiac conduction could influence in vivo cardiac morphogenesis independent of contractile forces. We show that the cardiac mutant dco(s226) develops heart failure and interrupted cardiac morphogenesis following uncoordinated ventricular contraction. Using in vivo optical mapping/calcium imaging, we determined that the dco cardiac phenotype was primarily due to aberrant ventricular conduction. Because cardiac contraction and intracardiac hemodynamic forces can also influence cardiac development, we further analyzed the dco phenotype in noncontractile hearts and observed that disorganized ventricular conduction could affect cardiomyocyte morphology and subsequent heart morphogenesis in the absence of contraction or flow. By positional cloning, we found that dco encodes Gja3/Cx46, a gap junction protein not previously implicated in heart formation or function. Detailed analysis of the mouse Cx46 mutant revealed the presence of cardiac conduction defects frequently associated with human heart failure. Overall, these in vivo studies indicate that cardiac electrical forces are required to preserve cardiac chamber morphology and may act as a key epigenetic factor in cardiac remodeling.

Live-cell imaging and analysis of actin-mediated mitochondrial fission.

Current approaches, such as fixed-cell imaging or single-snapshot imaging, are insufficient to capture cytoskeleton-mediated mitochondrial fission. Here, we present a protocol to capture actin-mediated mitochondrial fission using high-resolution time-lapse imaging. We describe steps starting from cell preparation and mitochondria labeling through to live-cell imaging and final analysis. This approach is also applicable for analysis of multiple cytoskeleton-mediated organelle events such as vesicle trafficking, membrane fusion, and endocytic events in live cells. For complete details on the use and execution of this protocol, please refer to Shimura et al. (2021).1.

Echocardiography Recording in Awake Miniature Pigs.

Echocardiography uses ultrasonic waves to non-invasively assess cardiac structure and function and is the standard of care for cardiac assessment and monitoring. The miniature pig, or minipig, is increasingly being used as a model of cardiac disease in medical research. Pigs are notoriously difficult to restrain and handle safely, and, therefore, research echocardiography in this species is almost always performed under anesthesia or heavy sedation. Anesthetics and sedatives universally affect cardiovascular function and may cause the depression of cardiac output and blood pressure, increases or decreases in heart rate and systemic vascular resistance, changes in the electrical rhythm, and altered coronary blood flow. Therefore, sedated or anesthetized echocardiography may not accurately depict the progression of cardiac disease in large animal models, thereby limiting the translational value of these important studies. This paper describes a novel device that allows for standing awake echocardiography in minipigs. In addition, training techniques used to teach pigs to tolerate this painless and non-invasive procedure without the need for hemodynamic-altering anesthetics are described. Standing awake echocardiography represents a safe and feasible way to perform the most common cardiac monitoring test in minipigs for cardiovascular research.