E R Simpson

Transient Regenerative Potential of the Neonatal Mouse Heart

The heart in a newborn mouse can rebuild itself after injury, but this regenerative capacity is lost within a few days. Certain fish and amphibians retain a robust capacity for cardiac regeneration throughout life, but the same is not true of the adult mammalian heart. Whether the capacity for cardiac regeneration is absent in mammals or whether it exists and is switched off early after birth has been unclear. We found that the hearts of 1-day-old neonatal mice can regenerate after partial surgical resection, but this capacity is lost by 7 days of age. This regenerative response in 1-day-old mice was characterized by cardiomyocyte proliferation with minimal hypertrophy or fibrosis, thereby distinguishing it from repair processes. Genetic fate mapping indicated that the majority of cardiomyocytes within the regenerated tissue originated from preexisting cardiomyocytes. Echocardiography performed 2 months after surgery revealed that the regenerated ventricular apex had normal systolic function. Thus, for a brief period after birth, the mammalian heart appears to have the capacity to regenerate.

Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family

We recently identified a brief time period during postnatal development when the mammalian heart retains significant regenerative potential after amputation of the ventricular apex. However, one major unresolved question is whether the neonatal mouse heart can also regenerate in response to myocardial ischemia, the most common antecedent of heart failure in humans. Here, we induced ischemic myocardial infarction (MI) in 1-d-old mice and found that this results in extensive myocardial necrosis and systolic dysfunction. Remarkably, the neonatal heart mounted a robust regenerative response, through proliferation of preexisting cardiomyocytes, resulting in full functional recovery within 21 d. Moreover, we show that the miR-15 family of microRNAs modulates neonatal heart regeneration through inhibition of postnatal cardiomyocyte proliferation. Finally, we demonstrate that inhibition of the miR-15 family from an early postnatal age until adulthood increases myocyte proliferation in the adult heart and improves left ventricular systolic function after adult MI. We conclude that the neonatal mammalian heart can regenerate after myocardial infarction through proliferation of preexisting cardiomyocytes and that the miR-15 family contributes to postnatal loss of cardiac regenerative capacity.

miR-15 Family Regulates Postnatal Mitotic Arrest of Cardiomyocytes

Rationale: Mammalian cardiomyocytes withdraw from the cell cycle during early postnatal development, which significantly limits the capacity of the adult mammalian heart to regenerate after injury. The regulatory mechanisms that govern cardiomyocyte cell cycle withdrawal and binucleation are poorly understood. Objective: Given the potential of microRNAs (miRNAs) to influence large gene networks and modify complex developmental and disease phenotypes, we searched for miRNAs that were regulated during the postnatal switch to terminal differentiation. Methods and Results: Microarray analysis revealed subsets of miRNAs that were upregulated or downregulated in cardiac ventricles from mice at 1 and 10 days of age (P1 and P10). Interestingly, miR-195 (a member of the miR-15 family) was the most highly upregulated miRNA during this period, with expression levels almost 6-fold higher in P10 ventricles relative to P1. Precocious overexpression of miR-195 in the embryonic heart was associated with ventricular hypoplasia and ventricular septal defects in &bgr;-myosin heavy chain–miR-195 transgenic mice. Using global gene profiling and argonaute-2 immunoprecipitation approaches, we showed that miR-195 regulates the expression of a number of cell cycle genes, including checkpoint kinase 1 (Chek1), which we identified as a highly conserved direct target of miR-195. Finally, we demonstrated that knockdown of the miR-15 family in neonatal mice with locked nucleic acid–modified anti-miRNAs was associated with an increased number of mitotic cardiomyocytes and derepression of Chek1. Conclusions: These findings suggest that upregulation of the miR-15 family during the neonatal period may be an important regulatory mechanism governing cardiomyocyte cell cycle withdrawal and binucleation.

Cytochromes P450 11: expression of the CYP19 (aromatase) gene: an unusual case of alternative promoter usage.

Family 19 of the P450 super family is responsible for the conversion of C19 androgenic steroids to the corresponding estrogens, a reaction known as aromatization because it involves conversion of the Δ4‐3‐one A‐ring of the androgens to the corresponding phenolic A‐ring characteristic of estrogens. The gene encoding human aromatase has been cloned and characterized and shown to be unusual compared to genes encoding other P450 enzymes, because there are numerous untranslated first exons that occur in aromatase transcripts in a tissue‐specific fashion due to differential splicing as a consequence of the use of tissue‐specific promoters. Thus, expression in the ovary uses a proximal promoter that is regulated primarily by cAMP. On the other hand, expression in the placenta uses a distal promoter located at least 40 kb upstream of the start of transcription that is regulated by retinoids. Other promoters are used in brain and adipose tissue. In the latter case, class I cytokines such as IL‐6 and IL‐11, as well as TNF‐α, are important regulatory factors. A common 3‘‐splice junction located upstream of the start of translation is used in all of the splicing events involved in the use of these various promoters. Thus, the coding region of the transcripts, and hence the protein, are identical regardless of the tissue site of expression; what differs in a tissue‐specific fashion is the 5‘‐end of the transcripts. This pattern of expression has great significance both from a phylogenetic and ontogenetic standpoint, as well as for the physiology and pathophysiology of estrogen formation, as will be discussed in thie review.—Simpson, E. R., Michael, M. D., Agarwal, V. R., Hinshelwood, M. M., Bulun, S. E., Zhao, T. Expression of the CYP19 (aromatase) gene: an unusual case of alternative promoter usage. FASEB J. 11, 29‐36 (1997)

Expression of P-450 enzyme activities in heterologous cells by transfection.

The product of the A gene of simian virus 40 (SV40), T antigen, is synthesized and accumulates in the nucleus of infected cells. This regulartory protein acts to initiate viral DNA replication as well as regulating SV40 gene transcription (Tjian 1981). Following transformation of CV-1 cells with an origin-defective mutant of SV40, the resultant monkey kidney cell line (called COS 1) produces T antigen (Gluzman 1981). Transfection of COS 1 cells with plasmid vectors which contain an SV40 origin of replication leads to replication of the plasmid DNA under the influence of T antigen. Accordingly the number of plasmid vectors is amplified in transfected COS 1 cells and the amplified plasmid DNA can then be transcribed leading to production of relatively large quantities of RNA derived from the plasmid. This RNA can, of course, be translated by the endogenous protein synthetic machinery in the COS 1 cells. When the plasmid vector contains a cDNA insert encoding a specific protein, readily detectable quantities of the protein encoded by the insert can be produced in COS 1 cells. A convenient vector for such expression studies is the pcD vector constructed by Okayama and Berg (1982) which contains an SV40 origin of replication and SV40 promotor sequence. A similar vector (pSVL) can be purchased from Pharmacia. pSVL contains a limited multiple cloning site which facilitates insertion of different cDNAs into this expression vector.

The miR-15 Family Regulates Post-natal Mitotic Arrest of Cardiomyocytes

Rationale: Mammalian cardiomyocytes withdraw from the cell cycle during early postnatal development, which significantly limits the capacity of the adult mammalian heart to regenerate after injury. The regulatory mechanisms that govern cardiomyocyte cell cycle withdrawal and binucleation are poorly understood. Objective: Given the potential of microRNAs (miRNAs) to influence large gene networks and modify complex developmental and disease phenotypes, we searched for miRNAs that were regulated during the postnatal switch to terminal differentiation. Methods and Results: Microarray analysis revealed subsets of miRNAs that were upregulated or downregulated in cardiac ventricles from mice at 1 and 10 days of age (P1 and P10). Interestingly, miR-195 (a member of the miR-15 family) was the most highly upregulated miRNA during this period, with expression levels almost 6-fold higher in P10 ventricles relative to P1. Precocious overexpression of miR-195 in the embryonic heart was associated with ventricular hypoplasia and ventricular septal defects in &bgr;-myosin heavy chain–miR-195 transgenic mice. Using global gene profiling and argonaute-2 immunoprecipitation approaches, we showed that miR-195 regulates the expression of a number of cell cycle genes, including checkpoint kinase 1 (Chek1), which we identified as a highly conserved direct target of miR-195. Finally, we demonstrated that knockdown of the miR-15 family in neonatal mice with locked nucleic acid–modified anti-miRNAs was associated with an increased number of mitotic cardiomyocytes and derepression of Chek1. Conclusions: These findings suggest that upregulation of the miR-15 family during the neonatal period may be an important regulatory mechanism governing cardiomyocyte cell cycle withdrawal and binucleation.

miR-15 Family Regulates Postnatal Mitotic Arrest of CardiomyocytesNovelty and Significance

Rationale: Mammalian cardiomyocytes withdraw from the cell cycle during early postnatal development, which significantly limits the capacity of the adult mammalian heart to regenerate after injury. The regulatory mechanisms that govern cardiomyocyte cell cycle withdrawal and binucleation are poorly understood. Objective: Given the potential of microRNAs (miRNAs) to influence large gene networks and modify complex developmental and disease phenotypes, we searched for miRNAs that were regulated during the postnatal switch to terminal differentiation. Methods and Results: Microarray analysis revealed subsets of miRNAs that were upregulated or downregulated in cardiac ventricles from mice at 1 and 10 days of age (P1 and P10). Interestingly, miR-195 (a member of the miR-15 family) was the most highly upregulated miRNA during this period, with expression levels almost 6-fold higher in P10 ventricles relative to P1. Precocious overexpression of miR-195 in the embryonic heart was associated with ventricular hypoplasia and ventricular septal defects in &bgr;-myosin heavy chain–miR-195 transgenic mice. Using global gene profiling and argonaute-2 immunoprecipitation approaches, we showed that miR-195 regulates the expression of a number of cell cycle genes, including checkpoint kinase 1 (Chek1), which we identified as a highly conserved direct target of miR-195. Finally, we demonstrated that knockdown of the miR-15 family in neonatal mice with locked nucleic acid–modified anti-miRNAs was associated with an increased number of mitotic cardiomyocytes and derepression of Chek1. Conclusions: These findings suggest that upregulation of the miR-15 family during the neonatal period may be an important regulatory mechanism governing cardiomyocyte cell cycle withdrawal and binucleation.