Human engineered heart tissue transplantation in a guinea pig chronic injury model.

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.

Deciphering the microRNA signature of pathological cardiac hypertrophy by engineered heart tissue- and sequencing-technology.

Pathological cardiac hypertrophy and fibrosis are modulated by a set of microRNAs, most of which have been detected in biologically complex animal models of hypertrophy by arrays with moderate sensitivity and disregard of passenger strand (previously "star") microRNAs. Here, we aimed at precisely analyzing the microRNA signature of cardiac hypertrophy and fibrosis by RNA sequencing in a standardized in vitro hypertrophy model based on engineered heart tissue (EHT). Spontaneously beating, force-generating fibrin EHTs from neonatal rat heart cells were subjected to afterload enhancement for 7days (AE-EHT), and EHTs without intervention served as controls. AE resulted in reduced contractile force and relaxation velocity, fibrotic changes and reactivation of the fetal gene program. Small RNAs were extracted from control and AE-EHTs and sequencing yielded almost 750 different mature microRNAs, many of which have never been described before in rats. The detection of both arms of the precursor stem-loop (pre-miRNA), namely -3p and -5p miRs, was frequent. 22 abundantly sequenced microRNAs were >1.3× upregulated and 15 abundantly sequenced microRNAs downregulated to <0.77×. Among the upregulated microRNAs were 3 pairs of guide and passenger strand microRNAs (miR-21-5p/-3p, miR-322-5p/-3p, miR-210-3p/-5p) and one single passenger strand microRNA (miR-140-3p). Among downregulated microRNAs were 3 pairs (miR-133a-3p/-5p, miR-30e-5p/3p, miR-30c-5p/-3p). Preincubating EHTs with anti-miR-21-5p markedly attenuated the AE-induced contractile failure, cardiomyocyte hypertrophy and fibrotic response, recapitulating prior results in whole animals. Taken together, AE-induced pathological hypertrophy in EHTs is associated with 37 differentially regulated microRNAs, including many passenger strands. Antagonizing miR-21-5p ameliorates dysfunction in this model.

Assessment of DNA synthesis in Islet-1⁺ cells in the adult murine heart.

RATIONALE: Islet-1 positive (Islet-1(+)) cardiac progenitor cells give rise to the right ventricle, atria and outflow tract during murine cardiac development. In the adult heart Islet-1 expression is limited to parasympathetic neurons, few cardiomyocytes, smooth muscle cells, within the proximal aorta and pulmonary artery and sinoatrial node cells. Its role in these cells is unknown. Here we tested the hypothesis that Islet-1(+) cells retain proliferative activity and may therefore play a role in regenerating specialized regions in the heart. METHODS AND RESULTS: DNA synthesis was analyzed by the incorporation of tritiated thymidine ((3)H-thymidine) in Isl-1-nLacZ mice, a transgenic model with an insertion of a nuclear beta-galactosidase in the Islet-1 locus. Mice received daily injections of (3)H-thymidine for 5 days. DNA synthesis was visualized throughout the heart by dipping autoradiography of cryosections. Colocalization of an nLacZ-signal and silver grains would indicate DNA synthesis in Islet-1(+) cells. Whereas Islet(-) non-myocyte nuclei were regularly marked by accumulation of silver grains, colocalization with nLacZ-signals was not detected in >25,000 cells analyzed. CONCLUSIONS: Islet-1(+) cells are quiescent in the adult heart, suggesting that, under normal conditions, even pacemaking cells do not proliferate at higher rates than normal cardiac myocytes.

Immature human engineered heart tissues engraft in a guinea pig chronic injury model.

Engineered heart tissue (EHT) transplantation represents an innovative, regenerative approach for heart failure patients. Late preclinical trials are underway, and a first clinical trial started recently. Preceding studies revealed functional recovery after implantation of in vitro-matured EHT in the subacute stage, whereas transplantation in a chronic injury setting was less efficient. When transplanting matured EHTs, we noticed that cardiomyocytes undergo a dedifferentiation step before eventually forming structured grafts. Therefore, we wanted to evaluate whether immature EHT (EHTIm) patches can be used for transplantation. Chronic myocardial injury was induced in a guinea pig model. EHTIm (15×106 cells) were transplanted within hours after casting. Cryo-injury led to large transmural scars amounting to 26% of the left ventricle. Grafts remuscularized 9% of the scar area on average. Echocardiographic analysis showed some evidence of improvement of left-ventricular function after EHTIm transplantation. In a small translational proof-of-concept study, human scale EHTIm patches (4.5×108 cells) were epicardially implanted on healthy pig hearts (n=2). In summary, we provide evidence that transplantation of EHTIm patches, i.e. without precultivation, is feasible, with similar engraftment results to those obtained using matured EHT.

Increased myofilament Ca2+ sensitivity and diastolic dysfunction as early consequences of Mybpc3 mutation in heterozygous knock-in mice.

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from gene mutations to the HCM phenotype remain incompletely understood, partially because current mouse models of HCM do not faithfully reflect the human situation and early hypertrophy confounds the interpretation of functional alterations. The goal of this study was to evaluate whether myofilament Ca(2+) sensitization and diastolic dysfunction are associated or precede the development of left ventricular hypertrophy (LVH) in HCM. We evaluated the function of skinned and intact cardiac myocytes, as well as the intact heart in a recently developed Mybpc3-targeted knock-in mouse model carrying a point mutation frequently associated with HCM. Compared to wild-type, 10-week old homozygous knock-in mice exhibited i) higher myofilament Ca(2+) sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca(2+) transient decay in intact myocytes, and iii) LVH, reduced fractional shortening, lower E/A and E'/A', and higher E/E' ratios by echocardiography and Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast, heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit LVH or systolic dysfunction, but exhibited higher myofilament Ca(2+) sensitivity, faster Ca(2+) transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca(2+) sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca(2+) transients point to compensatory mechanisms directed towards normalization of relaxation. We propose that HCM is a model for diastolic heart failure and this mouse model could be valuable in studying mechanisms and treatment modalities.

Piezo2 is not an indispensable mechanosensor in murine cardiomyocytes.

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.

No effect of thymosin beta-4 on the expression of the transcription factor Islet-1 in the adult murine heart.

The transcription factor Islet-1 marks a progenitor cell population of the second heart field during cardiogenesis. In the adult heart Islet-1 expression is limited to the sinoatrial node, the ventricular outflow tract, and parasympathetic ganglia. The regenerative effect in the injured mouse ventricle of thymosin beta-4 (TB4), a 43-aminoacid peptide, was associated with increased Islet-1 immunostaining, suggesting the induction of an Islet-1-positive progenitor state by TB4. Here we aimed to reassess this effect in a genetic model. Mice from the reporter mouse line Isl1-nLacZ were primed with TB4 and subsequently underwent myocardial infarction. Islet-1 expression was assessed 2, 7, and 14 days after infarction. We detected only a single Islet-1+ cell in 8 TB4 treated and infarcted hearts which located outside of the sinoatrial node, the outflow tract or cardiac ganglia (in ~2500 sections). Two cells were identified in 5 control infarcted hearts. TB4 did not induce LacZ positivity in ventricular explants cultures of Isl1-nLacZ mice nor did it affect the density of LacZ+ cells in explant cultures of nLacZ+ regions of the heart. In summary, we found no evidence that TB4 reactivates Islet-1 expression in adult mouse ventricle.

Cardiac repair in guinea pigs with human engineered heart tissue from induced pluripotent stem cells.

Myocardial injury results in a loss of contractile tissue mass that, in the absence of efficient regeneration, is essentially irreversible. Transplantation of human pluripotent stem cell-derived cardiomyocytes has beneficial but variable effects. We created human engineered heart tissue (hEHT) strips from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and hiPSC-derived endothelial cells. The hEHTs were transplanted onto large defects (22% of the left ventricular wall, 35% decline in left ventricular function) of guinea pig hearts 7 days after cryoinjury, and the results were compared with those obtained with human endothelial cell patches (hEETs) or cell-free patches. Twenty-eight days after transplantation, the hearts repaired with hEHT strips exhibited, within the scar, human heart muscle grafts, which had remuscularized 12% of the infarct area. These grafts showed cardiomyocyte proliferation, vascularization, and evidence for electrical coupling to the intact heart tissue in a subset of engrafted hearts. hEHT strips improved left ventricular function by 31% compared to that before implantation, whereas the hEET or cell-free patches had no effect. Together, our study demonstrates that three-dimensional human heart muscle constructs can repair the injured heart.

Cerebral expression of the alpha2-subunit of soluble guanylyl cyclase is linked to cerebral maturation and sensory pathway refinement during postnatal development.

Soluble guanylyl cylase (sGC) has been identified for being a receptor for the gaseous transmitters nitric oxide and carbon monoxide. Currently four subunits alpha1, alpha2, beta1, and beta2 have been characterized. Heterodimers of alpha and beta-subunits as well as homodimers of the beta2-subunit are known to constitute functional sGC which use GTP to form cGMP a potent signal molecule in a multitude of second messenger cascades. Since NO-cGMP signaling plays a pivotal role in neuronal development we analyzed the maturational expression pattern of the newly characterized alpha2-subunit of sGC within the brain of Wistar rats by means of RNase protection assay and immunohistochemistry. alpha2-subunit mRNA as well as immunoreactive alpha2-protein increased during postnatal cerebral development. Topographical analysis revealed a selective high expression of the alpha2-subunit in the choroid plexus and within developing sensory systems involving the olfactory and somatosensory system of the forebrain as well as parts of the auditory and visual system within the hindbrain. In cultured cortical neurons the alpha2-subunit was localized to the cell membrane, especially along neuronal processes. During the first 11 days of postnatal development several cerebral regions showed a distinct expression of the alpha2-subunit which was not paralleled by the alpha1/beta1-subunits especially within the developing thalamo-cortical circuitries of the somatosensory system. However, at later developmental stages all three subunits became more homogenously distributed among most cerebral regions, indicating that functional alpha1/beta1 and alpha2/beta1 heterodimers of sGC could be formed. Our findings indicate that the alpha2-subunit is an essential developmentally regulated constituent of cerebral sensory systems during maturation. In addition the alpha2-subunit may serve other functions than forming a functional heterodimer of sGC during the early phases of sensory pathway refinement.

Decreased phosphorylation levels of cardiac myosin-binding protein-C in human and experimental heart failure.

Cardiac myosin-binding protein-C (cMyBP-C) is an important regulator of cardiac contractility, and its phosphorylation by PKA is a mechanism that contributes to increased cardiac output in response to beta-adrenergic stimulation. It is presently unknown whether heart failure alters cMyBP-C phosphorylation. The present study determined the level of phosphorylated cMyBP-C in failing human hearts and in a canine model of pacing-induced heart failure. A polyclonal antibody directed against the major phosphorylation site of cMyBP-C (Ser-282) was generated and its specificity was confirmed by PKA phosphorylation with isoprenaline in cardiomyocytes and Langendorff-perfused mouse hearts. Left ventricular myocardial tissue from (i) patients with terminal heart failure (hHF; n=12) and nonfailing donor hearts (hNF; n=6) and (ii) dogs with rapid-pacing-induced end-stage heart failure (dHF; n=10) and sham-operated controls (dNF; n=10) were used for quantification of total cMyBP-C and phospho-cMyBP-C by Western blotting. Total cMyBP-C protein levels were similar in hHF and hNF as well as in dHF and dNF. In contrast, the ratio of phospho-cMyBP-C to total cMyBP-C levels were >50% reduced in hHF and >40% reduced in dHF. In summary, cMyBP-C phosphorylation levels are markedly decreased in human and experimental heart failure. Thus, the compromised contractile function of the failing heart might be in part attributable to reduced cMyBP-C phosphorylation levels.