Emma Simpson

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.

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.

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 β-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.

There was no significant difference in volume of semen produced by purebred Southdown rams kept in an air-conditioned room at 45–48° F. during the summer months when compared with rams kept at uncontrolled environmental conditions. From August 20 to September 24 the average motility rating for weekly collections was 70.3% motile cells for treated rams (air conditioned room) and 41.8% for control rams. For the same period, semen from treated rams contained 6.4% morphologically abnormal cells and the control rams 36.9%. Both of these differences were highly significant. The treated rams also had a significantly higher sperm cell concentration (3.4 vs. 2.4 million cells per mm3); their average rectal temperature and pulse rate also was significantly lower. Fifty ova were recovered from 30 ewes which were slaughtered after being bred early in the breeding season with semen from the control rams, and 26.0% of the ova were cleaved. Of 53 ova recovered from 30 ewes bred by the treated rams 64.2% were cleaved. Lambing percentages of similar groups of ewes not slaughtered were 13.3% and 50.0% for the control and treated rams, respectively. The differences between the ram groups in percent of ova fertilized and in percent of ewes lambing were both highly significant. Estimated embryonic death loss was higher (not significant) in ewes bred to control rams. Embryonic death rate was calculated to be 69.2% and 41.2% for ewes bred to control and treated rams, respectively. Estimated percentage of embryonic death loss in ewes bred to individual rams ranged from 14.3 to 100, with an over-all estimated embryonic loss of 48.0%. These results indicate that summer temperatures are partly responsible for poor conception rate of ewes bred to Southdown rams early in the breeding season. They also point out the possibility of improving conception rate early in the breeding season by keeping rams at lower environmental temperatures during the summer months.

Thirty-five dark-faced, crossbred Western ewes were fed 50 mg. of 6-methyl-17-acetoxyprogesterone (MAP) daily for 14 days. Two ewes came into estrus on the fifth day of treatment, and there was evidence that another ewe had ovulated during treatment without showing estrus. Twenty-six ewes (74.3%) came into estrus within 4 days and 29 ewes (82.8%) within 8 days post-treatment (av., 3.14 days). Ovulation rate in 10 ewes slaughtered for fertility data was 1.27 and 70% of the ova were cleaved. Of 19 ewes retained for lambing data, 9 returned to estrus in 15 to 17 days (av., 16.4 days) and 8 (42.1%) lambed to breeding at the first post-treatment estrus. Forty ewes were used in a factorial design in which four groups of 10 ewes each received 60 or 90 mg. of MAP for 15 or 18 days. None of these ewes came into estrus during treatment. Thirty-eight (95.0%) of these ewes came in heat 2 to 5 days (av., 3.2 days) post-treatment. The ovulation rate for 19 ewes slaughtered was 1.42, and 63% of the ova were cleaved. Sixteen of 19 ewes (84.2%) retained for lambing data lambed to breeding at the first post-treatment estrus. In the two trials 89.3% of the ewes returned to heat within 8 days (av., 3.16 days) after the end of treatment. Sixty-three percent lambed after breeding at the first post-treatment estrus, and 84.2% after breeding at the second post-treatment estrus. The variance in onset of post-treatment estrus was significantly lower than that for pre-treatment estrus (1.53 vs. 20.22). In Trial II the interval from end of treatment to estrus was significantly longer for the 90-mg. group (2.7 vs. 3.6 days) and for the 18-day group (2.9 vs. 3.6 days).