Cardiomyocytes stimulate angiogenesis after ischemic injury in a ZEB2-dependent manner

The disruption in blood supply due to myocardial infarction is a critical determinant for infarct size and subsequent deterioration in function. The identification of factors that enhance cardiac repair by the restoration of the vascular network is, therefore, of great significance. Here, we show that the transcription factor Zinc finger E-box-binding homeobox 2 (ZEB2) is increased in stressed cardiomyocytes and induces a cardioprotective cross-talk between cardiomyocytes and endothelial cells to enhance angiogenesis after ischemia. Single-cell sequencing indicates ZEB2 to be enriched in injured cardiomyocytes. Cardiomyocyte-specific deletion of ZEB2 results in impaired cardiac contractility and infarct healing post-myocardial infarction (post-MI), while cardiomyocyte-specific ZEB2 overexpression improves cardiomyocyte survival and cardiac function. We identified Thymosin β4 (TMSB4) and Prothymosin α (PTMA) as main paracrine factors released from cardiomyocytes to stimulate angiogenesis by enhancing endothelial cell migration, and whose regulation is validated in our in vivo models. Therapeutic delivery of ZEB2 to cardiomyocytes in the infarcted heart induces the expression of TMSB4 and PTMA, which enhances angiogenesis and prevents cardiac dysfunction. These findings reveal ZEB2 as a beneficial factor during ischemic injury, which may hold promise for the identification of new therapies

TECRL, a new life‐threatening inherited arrhythmia gene associated with overlapping clinical features of both LQTS and CPVT

Genetic causes of many familial arrhythmia syndromes remain elusive. In this study, whole-exome sequencing (WES) was carried out on patients from three different families that presented with life-threatening arrhythmias and high risk of sudden cardiac death (SCD). Two French Canadian probands carried identical homozygous rare variant in TECRL gene (p.Arg196Gln), which encodes the trans-2,3-enoyl-CoA reductase-like protein. Both patients had cardiac arrest, stress-induced atrial and ventricular tachycardia, and QT prolongation on adrenergic stimulation. A third patient from a consanguineous Sudanese family diagnosed with catecholaminergic polymorphic ventricular tachycardia (CPVT) had a homozygous splice site mutation (c.331+1G>A) in TECRL Analysis of intracellular calcium ([Ca(2+)]i) dynamics in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated from this individual (TECRLHom-hiPSCs), his heterozygous but clinically asymptomatic father (TECRLHet-hiPSCs), and a healthy individual (CTRL-hiPSCs) from the same Sudanese family, revealed smaller [Ca(2+)]i transient amplitudes as well as elevated diastolic [Ca(2+)]i in TECRLHom-hiPSC-CMs compared with CTRL-hiPSC-CMs. The [Ca(2+)]i transient also rose markedly slower and contained lower sarcoplasmic reticulum (SR) calcium stores, evidenced by the decreased magnitude of caffeine-induced [Ca(2+)]i transients. In addition, the decay phase of the [Ca(2+)]i transient was slower in TECRLHom-hiPSC-CMs due to decreased SERCA and NCX activities. Furthermore, TECRLHom-hiPSC-CMs showed prolonged action potentials (APs) compared with CTRL-hiPSC-CMs. TECRL knockdown in control human embryonic stem cell-derived CMs (hESC-CMs) also resulted in significantly longer APs. Moreover, stimulation by noradrenaline (NA) significantly increased the propensity for triggered activity based on delayed afterdepolarizations (DADs) in TECRLHom-hiPSC-CMs and treatment with flecainide, a class Ic antiarrhythmic drug, significantly reduced the triggered activity in these cells. In summary, we report that mutations in TECRL are associated with inherited arrhythmias characterized by clinical features of both LQTS and CPVT Patient-specific hiPSC-CMs recapitulated salient features of the clinical phenotype and provide a platform for drug screening evidenced by initial identification of flecainide as a potential therapeutic. These findings have implications for diagnosis and treatment of inherited cardiac arrhythmias

Z-disc protein CHAPb induces cardiomyopathy and contractile dysfunction in the postnatal heart

The Z-disc is a crucial structure of the sarcomere and is implicated in mechanosensation/transduction. Dysregulation of Z-disc proteins often result in cardiomyopathy. We have previously shown that the Z-disc protein Cytoskeletal Heart-enriched Actin-associated Protein (CHAP) is essential for cardiac and skeletal muscle development. Furthermore, the CHAP gene has been associated with atrial fibrillation in humans. Here, we studied the misregulated expression of CHAP isoforms in heart disease. Mice that underwent transverse aortic constriction and calcineurin transgenic (Tg) mice, both models of experimental heart failure, displayed a significant increase in cardiac expression of fetal isoform CHAPb. To investigate whether increased expression of CHAPb postnatally is sufficient to induce cardiomyopathy, we generated CHAPb Tg mice under the control of the cardiac-specific αMHC promoter. CHAPb Tg mice displayed cardiac hypertrophy, interstitial fibrosis and enlargement of the left atrium at three months, which was more pronounced at the age of six months. Hypertrophy and fibrosis were confirmed by evidence of activation of the hypertrophic gene program (Nppa, Nppb, Myh7) and increased collagen expression, respectively. Connexin40 and 43 were downregulated in the left atrium, which was associated with delayed atrioventricular conduction. Tg hearts displayed both systolic and diastolic dysfunction partly caused by impaired sarcomere function evident from a reduced force generating capacity of single cardiomyocytes. This co-incided with activation of the actin signalling pathway leading to the formation of stress fibers. This study demonstrated that the fetal isoform CHAPb initiates progression towards cardiac hypertrophy, which is accompanied by delayed atrioventricular conduction and diastolic dysfunction. Moreover, CHAP may be a novel therapeutic target or candidate gene for screening in cardiomyopathies and atrial fibrillatio

Sarcosin (Krp1) in skeletal muscle differentiation: gene expression profiling and knockdown experiments

SARCOSIN, also named Krp1, has been identified as a protein exclusively expressed in striated muscle tissue. Here we report on the role of SARCOSIN in skeletal muscle development and differentiation. We demonstrate, by means of whole-mount in situ hybridization, that Sarcosin mRNA is expressed in the myotome part of the mature somites in mouse embryos from embryonic day 9.5 onwards. Sarcosin is not expressed in the developing heart at these embryonic stages, and in adult tissues the mRNA expression levels are five times lower in the heart than in skeletal muscle. SARCOSIN protein partially co-localizes with the M-band protein myomesin and between and below laterally fusing myofibrils in adult skeletal muscle tissue. RNA interference mediated knock-down of SARCOSIN in the C2C12 myoblast cell line appeared to be stimulatory in the early phase of differentiation, but inhibitory at a later phase of differentiatio

Single-cell transcriptomics following ischemic injury identifies a role for B2M in cardiac repair

The efficiency of the repair process following ischemic cardiac injury is a crucial determinant for the progression into heart failure and is controlled by both intra- and intercellular signaling within the heart. An enhanced understanding of this complex interplay will enable better exploitation of these mechanisms for therapeutic use. We used single-cell transcriptomics to collect gene expression data of all main cardiac cell types at different time-points after ischemic injury. These data unveiled cellular and transcriptional heterogeneity and changes in cellular function during cardiac remodeling. Furthermore, we established potential intercellular communication networks after ischemic injury. Follow up experiments confirmed that cardiomyocytes express and secrete elevated levels of beta-2 microglobulin in response to ischemic damage, which can activate fibroblasts in a paracrine manner. Collectively, our data indicate phase-specific changes in cellular heterogeneity during different stages of cardiac remodeling and allow for the identification of therapeutic targets relevant for cardiac repair

Sarcomeric organization is disturbed and actin signalling is increased in CHAPb Tg hearts.

<p>(A) Immunostaining for CHAP (green) and α-actinin (red) show stress fiber formation (white arrows) in CHAP Tg hearts (B) Electron microscopy analysis of wt and CHAPb Tg hearts at 6 months of age. In CHAPb Tg the sarcomeres were irregular and Z-discs and intercalated discs (black arrow heads) are disorganized, while M-bands are absent (white arrow heads) (C) Wt and CHAPb Tg hearts at 6 months of age stained for RhoA (red). In wt mice RhoA is localized at the membrane of cardiomyocytes and shows a sarcomeric expression pattern. In CHAPb Tg hearts sarcomeric expression of RhoA is absent and membrane expression is increased. (D) Western blot analysis of 2 wt and 3 CHAPb Tg hearts at 6 months of age for RhoA (24kDa), α-actinin (100kDa), actin (42kDa), Ezrin(80 kDa)/moesin(80 kDa)/radixin (75 kDa; ERM), cofilin (19kDa), SRF (40 – 67kDa) and MEF2 (40-65kDa). GAPDH (38kDa) is used as loading control. (E) Working model: in adult wt mice CHAPa is localized at the Z-disc, leading to abundance of monomeric G-actin and subsequent low expression of SRF target genes. In CHAPb Tg mice CHAPb expression results in activation of RhoA, leading to a shift from G-actin to F-actin, binding of co-factors to SRF and activation of SRF target genes, such as ANF, BNP and β-MHC. Scale bars 20 μm in A and C, 1 μm in B.</p

CHAPb Tg mice show decrease cardiac performance.

<p><b>(</b>A) Representative 4-chamber view MRI images of wt and CHAPb Tg heart. Enlarged LA in CHAPb Tg is indicated with *. (B-G) MRI measurements of the left ventricle of wt (n = 4, white bars) and CHAPb Tg (n = 5, black bars) animals at 6 months of age. ED volume (B), ES volume (C), ejection fraction (D), cardiac output (E), LV mass ED (F) and LV mass ES (G). (H) Experimental setup for single membrane-permeabilized cardiomyocyte measurements to determine sarcomere function. Sarcomere force measurements at one month of age showed no difference in passive force (I; Fpas), while maximum force (J; Fmax) and Ca²⁺-sensitivity (K; pCa₅₀) were reduced in CHAPb Tg (n = 11, black bars) compared to wt (n = 12, white bars). ED (end diastolic), ES (end systolic) and LV (left ventricular). <i>t</i>-test: *, p<0.05; **, p<0.01; ***, p<0.001.</p

Hypertrophy and left atrial enlargement in CHAP Tg hearts.

<p>(A-C) Wt (left panels) and CHAPb Tg (right panels) at 3 and 6 months of age. (A) HE stained overview section. The left atrium in CHAPb Tg hearts is enlarged (indicated by *) compared to wt litter mates. (B) Higher magnification of left ventricle. In the left ventricle of CHAPb Tg hearts the cardiomyocytes are hypertrophic. (C) Sirius red staining of the left ventricle showing increase in interstitial fibrosis in CHAPb Tg. (D) Myocardial volume of the left atrium (left panels) and right atrium (right panels) in wt (white bars) and CHAPb Tg (black bars) hearts at 3 months of age (wt n = 4, Tg n = 4, T-test p< 0.001). (E) CHAPb Tg heart with severe phenotype showing pronounced atrial enlargement, filled by a thrombus and thickening of the ventricles. Scale bars 1 mm in A and D, 50 μm in B, C.</p

Chap is upregulated in mouse models of pathological cardiac hypertrophy.

<p>(A) Quantitative RT-PCR showing increased mRNA levels of both ChapA and B in hearts of transgenic mice expressing constitutively active calcineurin A (CnA Tg) (WT: n = 5, CnA Tg: n = 6; Students <i>t-test</i>: CnA Tg vs WT). (B) Western blot for CHAPa and CHAPb in adult wildtype hearts CnA Tg hearts (WT: n = 5, CnA Tg: n = 6). In wildtype hearts, CHAPa is the dominant isoform while in CnA Tg hearts both CHAPa and b were significantly upregulated. (C) Quantitative RT-PCR for ChapA showing mice subjected to transverse aortic constriction (TAC) for 1 day, 1 week and 3 weeks (n = 4, n = 4, and n = 10 respectively), with no significant regulation of ChapA mRNA compared to sham controls (n = 3, n = 3 and n = 7 respectively). (D) Quantitative RT-PCR for ChapB showing mice subjected to TAC with significant upregulation of ChapB mRNA (number of mice are same as in C). Gapdh was used as internal control in all experiments. <i>T-test</i> with Welch’s correction TAC vs sham: *, p<0.05; **, p<0.01;***, p<0.001.</p

Expression of hypertrophy markers and collagens in left ventricle.

<p>qPCR analysis showing mRNA expression of <i>Nppa</i> (A), <i>Nppb</i> (B), <i>Myh7</i> (C), <i>CollagenI</i> (D) <i>CollagenIII</i> (E), <i>Serca2</i> (F), <i>ChapA</i> (G), <i>ChapB</i> (H) and endogenous <i>ChapB</i> (I) in the left ventricle of wt (white bars) and CHAPb Tg (black bars) mice. <i>Gapdh</i>, <i>Pgk</i> and <i>H2a</i> were used as internal controls. (wt n = 3, Tg n = 3, T-test: *, p<0.05; **,p<0.01;***, p<0.001).</p