Guinea pigs have been a standard model in cardiovascular pharmacology and physiology research, but the advent of transgenic models has largely replaced them with mouse and rat models. However, guinea pigs remain important models in cardiac electrophysiology, drug-induced arrhythmias, or atherosclerosis research, and they have recently gained importance for studying one specific research question, that is, transplantation of pluripotent stem cell derived cardiomyocytes to repair the cryo-injured heart. Their human-like cardiac electrophysiology, together with their small size that facilitates handling and housing, make guinea pigs a valuable experimental model for these studies. However, repeated open heart surgeries in guinea pigs are technically demanding and accompanied by high mortality. In this study, we retrospectively examined sequential protocol modifications and describe how protocol refinements led to improved survival rates. Cryo-injury was performed in female Dunkin-Hartley guinea pigs under general anesthesia with a liquid nitrogen-cooled probe via a lateral thoracotomy. Cells were transplanted during a second surgery 7 days later. We analyzed data from up to 558 animals to determine mortality rates and morphologic and functional parameters. Initial studies revealed a mortality rate of ∼50%. Sequential modifications led to a significant reduction, with the refined protocol achieving a perioperative mortality rate of ∼30%. The procedures were completed in <35 minutes, and survival rates for the observation period (up to 8 weeks) were 70%. Scar size was evaluated in 144 (4 weeks, n = 92; 8 weeks, n = 52) animals and showed a significant, but shallow correlation with echocardiographically determined heart function. Taken together, refined surgery protocols allow safe and reproducible cryo-injury with subsequent cell injections in guinea pigs with an improved mortality rate.

Rationale: Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder caused by sarcomeric gene variants and associated with left ventricular (LV) hypertrophy and diastolic dysfunction. The role of the microtubule network has recently gained interest with the findings that -α-tubulin detyrosination (dTyr-tub) is markedly elevated in heart failure. Acute reduction of dTyr-tub by inhibition of the detyrosinase (VASH/SVBP complex) or activation of the tyrosinase (tubulin tyrosine ligase, TTL) markedly improved contractility and reduced stiffness in human failing cardiomyocytes, and thus poses a new perspective for HCM treatment. Objective: In this study, we tested the impact of chronic tubulin tyrosination in a HCM mouse model ( Mybpc3 -knock-in; KI), in human HCM cardiomyocytes and in SVBP-deficient human engineered heart tissues (EHTs). Methods and Results: AAV9-mediated TTL transfer was applied in neonatal wild-type (WT) rodents and 3-week-old KI mice and in HCM human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. We show that i) TTL for 6 weeks dose-dependently reduced dTyr-tub and improved contractility without affecting cytosolic calcium transients in WT cardiomyocytes; ii) TTL for 12 weeks improved diastolic filling, cardiac output and stroke volume and reduced stiffness in KI mice; iii) TTL for 10 days normalized cell hypertrophy in HCM hiPSC-cardiomyocytes; iv) TTL induced a marked transcription and translation of several tubulins and modulated mRNA or protein levels of components of mitochondria, Z-disc, ribosome, intercalated disc, lysosome and cytoskeleton in KI mice; v) SVBP-deficient EHTs exhibited reduced dTyr-tub levels, higher force and faster relaxation than TTL-deficient and WT EHTs. RNA-seq and mass spectrometry analysis revealed distinct enrichment of cardiomyocyte components and pathways in SVBP-KO vs. TTL-KO EHTs. Conclusion: This study provides the first proof-of-concept that chronic activation of tubulin tyrosination in HCM mice and in human EHTs improves heart function and holds promise for targeting the non-sarcomeric cytoskeleton in heart disease.

Aims Gene therapy with cardiac phosphodiesterases (PDEs), such as phosphodiesterase 4B (PDE4B), has recently been described to effectively prevent heart failure (HF) in mice. However, exact molecular mechanisms of its beneficial effects, apart from general lowering of cardiomyocyte cyclic adenosine monophosphate (cAMP) levels, have not been elucidated. Here, we studied whether gene therapy with two types of PDEs, namely PDE2A and PDE4B, can prevent pressure-overload-induced HF in mice by acting on and restoring altered cAMP compartmentation in distinct subcellular microdomains.Methods and results HF was induced by transverse aortic constriction followed by tail-vein injection of adeno-associated-virus type 9 vectors to overexpress PDE2A3, PDE4B3, or luciferase for 8 weeks. Heart morphology and function was assessed by echocardiography and histology which showed that PDE2A and especially PDE4B gene therapy could attenuate cardiac hypertrophy, fibrosis, and decline of contractile function. Live cell imaging using targeted cAMP biosensors showed that PDE overexpression restored altered cAMP compartmentation in microdomains associated with ryanodine receptor type 2 (RyR2) and caveolin-rich plasma membrane. This was accompanied by ameliorated caveolin-3 decline after PDE2A3 overexpression, reduced RyR2 phosphorylation in PDE4B3 overexpressing hearts, and antiarrhythmic effects of both PDEs measured under isoproterenol stimulation in single cells. Strong association of overexpressed PDE4B but not PDE2A with RyR2 microdomain could prevent calcium leak and arrhythmias in human-induced pluripotent stem-derived cardiomyocytes with the A2254V mutation in RyR2 causing catecholaminergic polymorphic ventricular tachycardia.Conclusion Our data indicate that gene therapy with phosphodiesterases can prevent HF including associated cardiac remodelling and arrhythmias by restoring altered cAMP compartmentation in functionally relevant subcellular microdomains. Graphical Abstract

Transplantation of pluripotent stem cell-derived cardiomyocytes is a novel promising cell-based therapeutic approach for patients with heart failure. However, engraftment arrhythmias are a predictable life-threatening complication and represent a major hurdle for clinical translation. Thus, we wanted to experimentally study whether impulse generation by transplanted cardiomyocytes can propagate to the host myocardium and overdrive the recipient rhythm. We transplanted human induced pluripotent stem cell-derived cardiomyocytes expressing the optogenetic actuator Bidirectional Pair of Opsins for Light-induced Excitation and Silencing (BiPOLES) in a guinea pig injury model. Eight weeks after transplantation ex vivo, Langendorff perfusion was used to assess electrical coupling. Pulsed photostimulation was applied to specifically activate the engrafted cardiomyocytes. Photostimulation resulted in ectopic pacemaking that propagated to the host myocardium, caused non-sustained arrhythmia, and stimulated the recipient heart with higher pacing frequency (4/9 hearts). Our study demonstrates that transplanted cardiomyocytes can (1) electrically couple to the host myocardium and (2) stimulate the recipient heart. Thus, our results provide experimental evidence for an important aspect of engraftment-induced arrhythmia induction and thereby support the current hypothesis that cardiomyocyte automaticity can serve as a trigger for ventricular arrhythmias.

BACKGROUND:Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder caused by sarcomeric gene variants and associated with left ventricular hypertrophy and diastolic dysfunction. The role of the microtubule network has recently gained interest with the findings that microtubule detyrosination (dTyr-MT) is markedly elevated in heart failure. Acute reduction of dTyr-MT by inhibition of the detyrosinase (VASH [vasohibin]/SVBP [small VASH-binding protein] complex) or activation of the tyrosinase (TTL [tubulin tyrosine ligase]) markedly improved contractility and reduced stiffness in human failing cardiomyocytes and thus posed a new perspective for HCM treatment. In this study, we tested the impact of chronic tubulin tyrosination in an HCM mouse model (Mybpc3 knock-in), in human HCM cardiomyocytes, and in SVBP-deficient human engineered heart tissues (EHTs).METHODS:Adeno-associated virus serotype 9-mediated TTL transfer was applied in neonatal wild-type rodents, in 3-week-old knock-in mice, and in HCM human induced pluripotent stem cell-derived cardiomyocytes.RESULTS:We show (1) TTL for 6 weeks dose dependently reduced dTyr-MT and improved contractility without affecting cytosolic calcium transients in wild-type cardiomyocytes; (2) TTL for 12 weeks reduced the abundance of dTyr-MT in the myocardium, improved diastolic filling, compliance, cardiac output, and stroke volume in knock-in mice; (3) TTL for 10 days normalized cell area in HCM human induced pluripotent stem cell-derived cardiomyocytes; (4) TTL overexpression activated transcription of tubulins and other cytoskeleton components but did not significantly impact the proteome in knock-in mice; (5) SVBP-deficient EHTs exhibited reduced dTyr-MT levels, higher force, and faster relaxation than TTL-deficient and wild-type EHTs. RNA sequencing and mass spectrometry analysis revealed distinct enrichment of cardiomyocyte components and pathways in SVBP-deficient versus TTL-deficient EHTs.CONCLUSIONS:This study provides the first proof of concept that chronic activation of tubulin tyrosination in HCM mice and in human EHTs improves heart function and holds promise for targeting the nonsarcomeric cytoskeleton in heart disease.

Rationale Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder caused by sarcomeric gene variants and associated with left ventricular (LV) hypertrophy and diastolic dysfunction. The role of the microtubule network has recently gained interest with the findings that α-tubulin detyrosination (dTyr-tub) is markedly elevated in heart failure. Reduction of dTyr-tub by inhibition of the detyrosinase (VASH/SVBP complex) or activation of the tyrosinase (tubulin tyrosine ligase, TTL) markedly improved contractility and reduced stiffness in human failing cardiomyocytes, and thus poses a new perspective for HCM treatment. Objective In this study, we tested the impact of targeting dTyr-tub in a mouse model of HCM, the Mybpc3 -targeted knock-in (KI) mice, and in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and engineered heart tissues (EHTs) deficient in SVBP or TTL. Methods and Results TTL gene transfer was tested in wild-type (WT) mice and rats and in adult KI mice. We show that i) TTL dose-dependently reduced dTyr-tub and improved contractility without affecting cytosolic calcium transients in WT cardiomyocytes; ii) TTL partially improved LV function and diastolic filling, reduced stiffness and normalized cardiac output and stroke volume in KI mice; iii) TTL induced a marked transcription and translation of several tubulins in KI mice; iv) TTL modulated mRNA or protein levels of components of mitochondria, Z-disc, ribosome, intercalated disc, lysosome and cytoskeleton in KI mice; v) SVBP-KO and TTL-KO EHTs exhibited low and high dTyr-tub levels, higher and lower force of contraction and enhanced and prolonged relaxation than in WT EHTs, respectively. RNA-seq and mass spectrometry analysis revealed distinct enrichment of cardiomyocyte components and pathways in SVBP-KO vs. TTL-KO EHTs. Conclusion This study provides evidence that reducing dTyr-tub improves function in HCM mouse hearts and human EHTs and holds promise for targeting the non-sarcomeric cytoskeleton in heart disease.

A short-term increase in ventricular filling leads to an immediate (Frank-Starling mechanism) and a slower (Anrep effect) rise in cardiac contractility, while long-term increased cardiac load (e.g., in arterial hypertension) decreases contractility. Whether these answers to mechanical tension are mediated by specific sensors in cardiomyocytes remains elusive. In this study, the piezo2 protein was evaluated as a potential mechanosensor. Piezo2 was found to be upregulated in various rat and mouse cardiac tissues upon mechanical or pharmacological stress. To investigate its function, C57BL/6J mice with homozygous cardiomyocyte-specific piezo2 knockout [Piezo2-KO] were created. To this end, α-MHC-Cre mice were crossed with homozygous “floxed” piezo2 mice. α-MHC-Cre mice crossed with wildtype mice served as controls [WT-Cre + ]. In cardiomyocytes of Piezo2-KO mice, piezo2 mRNA was reduced by > 90% and piezo2 protein was not detectable. Piezo2-KO mice displayed no morphological abnormalities or altered cardiac function under nonstressed conditions. In a subsequent step, hearts of Piezo2-KO or WT-Cre + -mice were stressed by either three weeks of increased afterload (angiotensin II, 2.5 mg/kg/day) or one week of hypercontractility (isoprenaline, 30 mg/kg/day). As expected, angiotensin II treatment in WT-Cre + -mice resulted in higher heart and lung weight (per body weight, + 38%, + 42%), lower ejection fraction and cardiac output (− 30%, − 39%) and higher left ventricular anterior and posterior wall thickness (+ 34%, + 37%), while isoprenaline led to higher heart weight (per body weight, + 25%) and higher heart rate and cardiac output (+ 24%, + 54%). The Piezo2-KO mice reacted similarly with the exception that the angiotensin II-induced increases in wall thickness were blunted and the isoprenaline-induced increase in cardiac output was slightly less pronounced. As cardiac function was neither severely affected under basal nor under stressed conditions in Piezo2-KO mice, we conclude that piezo2 is not an indispensable mechanosensor in cardiomyocytes.

Background: Transplantation of pluripotent stem cell–derived cardiomyocytes represents a promising therapeutic strategy for cardiac regeneration, and the first clinical studies in patients with heart failure have commenced. Yet, little is known about the mechanism of action underlying graft-induced benefits. Here, we explored whether transplanted cardiomyocytes actively contribute to heart function. Methods: We injected cardiomyocytes with an optogenetic off-on switch in a guinea pig cardiac injury model. Results: Light-induced inhibition of engrafted cardiomyocyte contractility resulted in a rapid decrease of left ventricular function in ≈50% (7/13) animals that was fully reversible with the offset of photostimulation. Conclusions: Our optogenetic approach demonstrates that transplanted cardiomyocytes can actively participate in heart function, supporting the hypothesis that the delivery of new force-generating myocardium can serve as a regenerative therapeutic strategy.

Myocardial injury leads to an irreversible loss of cardiomyocytes (CM). The implantation of human engineered heart tissue (EHT) has become a promising regenerative approach. Previous studies exhibited beneficial, dose-dependent effects of human induced pluripotent stem cell (hiPSC)-derived EHT patch transplantation in a guinea pig model in the subacute phase of myocardial injury. Yet, advanced heart failure often results from a chronic remodeling process. Therefore, from a clinical standpoint it is worthwhile to explore the ability to repair the chronically injured heart. In this study human EHT patches were generated from hiPSC-derived CMs (15 × 106 cells) and implanted epicardially four weeks after injury in a guinea pig cryo-injury model. Cardiac function was evaluated by echocardiography after a follow-up period of four weeks. Hearts revealed large transmural myocardial injuries amounting to 27% of the left ventricle. EHT recipient hearts demonstrated compact muscle islands of human origin in the scar region, as indicated by a positive staining for human Ku80 and dystrophin, remuscularizing 5% of the scar area. Echocardiographic analysis demonstrated no significant functional difference between animals that received EHT patches and animals in the cell-free control group (fractional area change 36% vs. 34%). Thus, EHT patches engrafted in the chronically injured heart but in contrast to the subacute model, grafts were smaller and EHT patch transplantation did not improve left ventricular function, highlighting the difficulties for a regenerative approach.

Abstract OBJECTIVES Univentricular malformations are severe cardiac lesions with limited therapeutic options and a poor long-term outcome. The staged surgical palliation (Fontan principle) results in a circulation in which venous return is conducted to the pulmonary arteries via passive laminar flow. We aimed to generate a contractile subpulmonary neo-ventricle from engineered heart tissue (EHT) to drive pulmonary flow actively. METHODS A three-dimensional tubular EHT (1.8-cm length, 6-mm inner diameter, ca. 1-mm wall thickness) was created by casting human-induced pluripotent stem cell-derived cardiomyocytes (0.9 ml, 18 mio/ml) embedded in a fibrin-based hydrogel around a silicone tube. EHTs were cultured under continuous, pulsatile flow through the silicone tube for 23 days. RESULTS The constructs started to beat macroscopically at days 8–14 and remained stable in size and shape over the whole culture period. Tubular EHTs showed a coherent beating pattern after 23 days in culture, and isovolumetric pressure measurements demonstrated a coherent pulsatile wave formation with an average frequency of 77 ± 5 beats/min and an average pressure of 0.2 mmHg. Histological analysis revealed cardiomyocytes mainly localized along the inner and outer curvature of the tubular wall with mainly longitudinal alignment. Cell density in the center of the tubular wall was lower. CONCLUSIONS A simple tube-shaped contractile EHT was generated from human-induced pluripotent stem cells and developed a synchronous beating pattern. Further steps need to focus on optimizing support materials, flow rates and geometry to obtain a construct that creates sufficient pressures to support a directed and pulsatile blood flow.

Background: Human engineered heart tissue (EHT) transplantation represents a potential regenerative strategy for patients with heart failure and has been successful in preclinical models. Clinical application requires upscaling, adaptation to good manufacturing practices, and determination of the effective dose. Methods: Cardiomyocytes were differentiated from 3 different human induced pluripotent stem cell lines including one reprogrammed under good manufacturing practice conditions. Protocols for human induced pluripotent stem cell expansion, cardiomyocyte differentiation, and EHT generation were adapted to substances available in good manufacturing practice quality. EHT geometry was modified to generate patches suitable for transplantation in a small-animal model and perspectively humans. Repair efficacy was evaluated at 3 doses in a cryo-injury guinea pig model. Human-scale patches were epicardially transplanted onto healthy hearts in pigs to assess technical feasibility. Results: We created mesh-structured tissue patches for transplantation in guinea pigs (1.5×2.5 cm, 9–15×10 6 cardiomyocytes) and pigs (5×7 cm, 450×10 6 cardiomyocytes). EHT patches coherently beat in culture and developed high force (mean 4.6 mN). Cardiomyocytes matured, aligned along the force lines, and demonstrated advanced sarcomeric structure and action potential characteristics closely resembling human ventricular tissue. EHT patches containing ≈4.5, 8.5, 12×10 6 , or no cells were transplanted 7 days after cryo-injury (n=18–19 per group). EHT transplantation resulted in a dose-dependent remuscularization (graft size: 0%–12% of the scar). Only high-dose patches improved left ventricular function (+8% absolute, +24% relative increase). The grafts showed time-dependent cardiomyocyte proliferation. Although standard EHT patches did not withstand transplantation in pigs, the human-scale patch enabled successful patch transplantation. Conclusions: EHT patch transplantation resulted in a partial remuscularization of the injured heart and improved left ventricular function in a dose-dependent manner in a guinea pig injury model. Human-scale patches were successfully transplanted in pigs in a proof-of-principle study.

The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the main proteolytic systems involved in cellular homeostasis. Since cardiomyocytes, as terminally differentiated cells, lack the ability to share damaged proteins with their daughter cells, they are especially reliant on these protein degradation systems for their proper function. Alterations of the UPS and ALP have been reported in a wide range of cardiac diseases, including cardiomyopathies. In this study, we determined whether the UPS and ALP are altered in a mouse model of eccentric left ventricular (LV) hypertrophy expressing both cyclin T1 and Gαq under the control of the cardiac-specific α-myosin heavy chain promoter (double transgenic; DTG). Compared to wild-type (WT) littermates, DTG mice showed higher end-diastolic (ED) LV wall thicknesses and diameter with preserved ejection fraction (EF). The cardiomyopathic phenotype was further confirmed by an upregulation of the fetal gene program and genes associated with fibrosis as well as a downregulation of genes involved in Ca2+ handling. Likewise, higher NT-proBNP levels were detected in DTG mice. Investigation of the UPS showed elevated steady-state levels of (poly)ubiquitinated proteins without alterations of all proteasomal activities in DTG mice. Evaluation of ALP key marker revealed a mixed pattern with higher protein levels of microtubule-associated protein 1 light chain 3 beta (LC3)-I and lysosomal-associated membrane protein-2, lower protein levels of beclin-1 and FYVE and coiled-coil domain-containing protein 1 (FYCO1) and unchanged protein levels of p62/SQSTM1 in DTG mice when compared to WT. At transcriptional level, a > 1.2-fold expression was observed for Erbb2, Hdac6, Lamp2, Nrg1, and Sqstm1, while a < 0.8-fold expression was revealed for Fyco1 in DTG mice. The results related to the ALP suggested overall a repression of the ALP during the initiation process, but an induction of the ALP at the level of autophagosome-lysosome fusion and the delivery of ubiquitinated cargo to the ALP for degradation.

3′,5′-Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which plays critical roles in cardiac function and disease. In adult mouse ventricular myocytes (AMVMs), several distinct functionally relevant microdomains with tightly compartmentalized cAMP signaling have been described. At least two types of microdomains reside in AMVM plasma membrane which are associated with caveolin-rich raft and non-raft sarcolemma, each with distinct cAMP dynamics and their differential regulation by receptors and cAMP degrading enzymes phosphodiesterases (PDEs). However, it is still unclear how cardiac disease such as hypertrophy leading to heart failure affects cAMP signals specifically in the non-raft membrane microdomains. To answer this question, we generated a novel transgenic mouse line expressing a highly sensitive Förster resonance energy transfer (FRET)-based biosensor E1-CAAX targeted to non-lipid raft membrane microdomains of AMVMs and subjected these mice to pressure overload induced cardiac hypertrophy. We could detect specific changes in PDE3-dependent compartmentation of β-adrenergic receptor induced cAMP in non-raft membrane microdomains which were clearly different from those occurring in caveolin-rich sarcolemma. This indicates differential regulation and distinct responses of these membrane microdomains to cardiac remodeling.

The current study describes the optimization of EHT geometry. EHT patches structurally and physiologically resembled adult human ventricular myocardium and EHT patch transplantation resulted in large human myocardial grafts that led to complete coverage of the scar area.

P90 ribosomal S6 kinases (RSK) are ubiquitously expressed and regulate responses to neurohumoral stimulation. To study the role of RSK signalling on cardiac myocyte function and protein phosphorylation, pharmacological RSK inhibitors were tested. Here, the ATP competitive N-terminal kinase domain-targeting compounds D1870 and SL0101 and the allosteric C-terminal kinase domain-targeting FMK were evaluated regarding their ability to modulate cardiac myocyte protein phosphorylation. Exposure to D1870 and SL0101 significantly enhanced phospholamban (PLN) Ser16 and cardiac troponin I (cTnI) Ser22/23 phosphorylation in response to D1870 and SL0101 upon exposure to phenylephrine (PE) that activates RSK. In contrast, FMK pretreatment significantly reduced phosphorylation of both proteins in response to PE. D1870-mediated enhancement of PLN Ser16 phosphorylation was also observed after exposure to isoprenaline or noradrenaline (NA) stimuli that do not activate RSK. Inhibition of β-adrenoceptors by atenolol or cAMP-dependent protein kinase (PKA) by H89 prevented the D1870-mediated increase in PLN phosphorylation, suggesting that PKA is the kinase responsible for the observed phosphorylation. Assessment of changes in cAMP formation by FRET measurements revealed increased cAMP formation in vicinity to PLN after exposure to D1870 and SL0101. D1870 inhibited phosphodiesterase activity similarly as established PDE inhibitors rolipram or 3-isobutyl-1-methylxanthine. Assessment of catecholamine-mediated force development in rat ventricular muscle strips revealed significantly reduced EC50 for NA after D1870 pretreatment (DMSO/NA: 2.33 μmol/L vs. D1870/NA: 1.30 μmol/L). The data reveal enhanced cardiac protein phosphorylation by D1870 and SL0101 that was not detectable in response to FMK. This disparate effect might be attributed to off-target inhibition of PDEs with impact on muscle function as demonstrated for D1870.

AIM:Muscarinic acetylcholine receptors (AChMR1-5) are fundamental for cellular responses upon release of the neurotransmitter acetylcholine (ACh) from parasympathetic nerve fibers. ACh is the prototypical agonist stimulating endothelium-dependent dilation, but most blood vessels lack parasympathetic innervation, raising the question as to the physiologic function of endothelial AChMR in vivo. Global deletion of AChM3R revealed a role in ACh-induced vasodilation in vitro and food uptake, but overall cardiovascular homeostasis has not been examined thoroughly.METHODS:To characterize the function of endothelial AChM3R in vivo, we deleted AChM3R specifically in endothelial cells with an inducible or a non-inducible Cre-loxP system, driven by the endothelium-specific promoters VE-cadherin (indEC-M3R-/- ) or TIE2 (tek2; EC-M3R-/- ) and examined arteriolar dilation in the cremaster microcirculation, arterial pressure and cardiac function in these mice in vivo.RESULTS:In both EC-M3R-/- , ACh-induced dilation was strongly impaired in arterioles in vivo, while responses to other dilators were mostly preserved. However, arterial pressure (indEC-M3R-/- ) and arteriolar tone as a surrogate for peripheral vascular resistance did not differ between EC-M3R-/- and control mice. Aged EC-M3R-/- mice (74-78 weeks) did not differ in body weight, heart weight, cardiac structure or contractile function from controls.CONCLUSION:We conclude that AChM3R elicits the endothelium-dependent dilation upon ACh also in arterioles in vivo. Despite this prominent role, the endothelial deletion of AChM3R does not affect overall cardiovascular homeostasis. Thus, their physiologic function in endothelial cells remains obscure.