Neurons regulate cardiomyocyte proliferation through clock genes.
Sympathetic neurons (SNs) regulate heart rate, conduction velocity, contractility and relaxation of the myocardium. Disruption of SNs in adult cardiac disease, leads to arrhythmias, myocardial dysfunction and sudden cardiac death. However, the role of SNs during cardiac development and whether disruption of cardiac SNs at embryonic stages is associated with disease progression in adult life is unknown. Moreover, controversy exists regarding the effect of SNs on neonatal myocardial regeneration after injury. As the heart is innervated by SNs from mid-gestation and the innervation continues through the neonatal stage, we generated genetically modified mice with profoundly reduced cardiac-specific sympathetic innervation starting at embryonic stages. Inhibition of sympathetic innervation resulted in larger heart size with increased number of myocytes that were smaller and more mononuclear. Analysis also confirmed increased cardiomyocyte proliferation. Interestingly, transcriptomic analysis of P7 and P14 hearts verified dysregulated cell cycle, calcium homeostasis and circadian genes. Remarkably, this led to persistently increased heart size and reduced function at adult stages. To investigate the mechanism whereby SNs, affect cardiomyocyte proliferation, we first co-cultured human stem cell-derived or mouse cardiomyocytes with ganglionic SNs and confirmed similar gene expression patterns. Moreover, alpha1 and beta2-specific adrenergic receptor agonists and norepinephrine, reduced cell cycle genes and myocyte proliferation and upregulated circadian genes such as Period1 and Period2. As circadian genes have been linked with regulation of cell cycle and apoptosis, we analyzed neonatal hearts from Period1/Period2 DKO mice and curiously discovered increased heart size, cardiomyocyte proliferation and cell cycle genes, and suppression of the Wee1 kinase an inhibitor of cell mitosis. More importantly, norepinephrine did not alter cardiomyocyte proliferation and cell cycle genes in Period1/Period2 DKO myocytes. To our knowledge this is the first study to provide direct evidence of the effect of SNs in the regulation of circadian genes in the heart and their role on neonatal cardiomyocyte proliferation.
Introduction: Alpha actinin-2 (ACTN2) is a major cytoskeletal protein that plays a critical role in maintaining the structural and functional integrity of the sarcomere. ACTN2 mutations, although rare, have been shown to be associated with various types of cardiomyopathy. Using genome-wide association and multi-omic approaches, we recently identified non-coding variants that showed a strong association with heart failure (HF). Two of the variants are within an evolutionary conserved transcriptional enhancer region that regulates the ACTN2 gene in human stem cell derived cardiomyocytes (hPSC-CMs). However, the role of cardiac enhancers in the development of HF remains unknown, therefore we used engineered heart tissues (EHT) from hPSC-CMs to investigate the effects of the ACTN2 enhancer. Methods: We used hPSC-CMs and performed morphologic and functional analyses by immunostaining, calcium transient measurements and quantitative assays such as western blotting, proteomics, qPCR and single cell transcriptomics. Next, we utilized CRISPR interference (CRISPRi) and chromatin immunoprecipitation (ChIP) to analyze the transcriptional regulation of the ACTN2 enhancer. Finally, we measured force generation using EHTs. Results: We first engineered hPSC-CMs carrying an ACTN2 enhancer deletion. This resulted in decreased ACTN2 gene and protein expression, and cardiomyocytes developed myofibrillar disarray, hypertrophy, lower beating rates and suppressed calcium transients. Moreover, EHTs demonstrated reduced mechanical force. Using CRISPRi and ChIP we found that the transcription factor MEF2c binds a short DNA sequence containing an enhancer variant to regulate ACTN2 expression. Single cell RNA-Seq analysis of hPSC-CMs treated with isoproterenol to model the hyperadrenergic state observed in HF, revealed the induction of pathways involved in protein quality control, actin fragmentation and apoptosis. We have also recently developed a mouse model to address the effect of the enhancer in vivo . Conclusions: Our study confirms that a conserved and clinically relevant enhancer region can effectively regulate ACTN2 , and variants within that region can have detrimental consequences on CMs which can contribute to HF.
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