Summary The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary post-natal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen rich postnatal environment is the upstream signal that results in cell cycle arrest of cardiomyocytes. Here we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, while hyperoxemia and ROS generators shorten it. These findings uncover a previously unrecognized protective mechanism that mediates cardiomyocyte cell cycle arrest in exchange for utilization of oxygen dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be important component of cardiomyocyte proliferation-based therapeutic approaches.

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.

Condit ME, Lefer DJ. The polysulfide diallyl trisulfide protects the ischemic myocardium by preservation of endogenous hydrogen sulfide and increasing nitric oxide bioavailability. Am J Physiol Heart Circ Physiol 302: H2410-H2418, 2012. First published March 30, 2012 doi:10.1152/ajpheart.00044.2012.-Diallyl trisulfide (DATS), a polysulfide constituent found in garlic oil, is capable of the release of hydrogen sulfide (H 2S). H2S is a known cardioprotective agent that protects the heart via antioxidant, antiapoptotic, anti-inflammatory, and mitochondrial actions. Here, we investigated DATS as a stable donor of H 2S during myocardial ischemiareperfusion (MI/R) injury in vivo. We investigated endogenous H 2S levels, infarct size, postischemic left ventricular function, mitochondrial respiration and coupling, endothelial nitric oxide (NO) synthase (eNOS) activation, and nuclear E2-related factor (Nrf2) translocation after DATS treatment. Mice were anesthetized and subjected to a surgical model of MI/R injury with and without DATS treatment (200 g/kg). Both circulating and myocardial H 2S levels were determined using chemiluminescent gas chromatography. Infarct size was measured after 45 min of ischemia and 24 h of reperfusion. Troponin I release was measured at 2, 4, and 24 h after reperfusion. Cardiac function was measured at baseline and 72 h after reperfusion by echocardiography. Cardiac mitochondria were isolated after MI/R, and mitochondrial respiration was investigated. NO metabolites, eNOS phosphorylation, and Nrf2 translocation were determined 30 min and 2 h after DATS administration. Myocardial H 2S levels markedly decreased after I/R injury but were rescued by DATS treatment (P Ͻ 0.05). DATS administration significantly reduced infarct size per area at risk and per left ventricular area compared with control (P Ͻ 0.001) as well as circulating troponin I levels at 4 and 24 h (P Ͻ 0.05). Myocardial contractile function was significantly better in DATS-treated hearts compared with vehicle treatment (P Ͻ 0.05) 72 h after reperfusion. DATS reduced mitochondrial respiration in a concentration-dependent manner and significantly improved mitochondrial coupling after reperfusion (P Ͻ 0.01). DATS activated eNOS (P Ͻ 0.05) and increased NO metabolites (P Ͻ 0.05). DATS did not appear to significantly induce the Nrf2 pathway. Taken together, these data suggest that DATS is a donor of H 2S that can be used as a cardioprotective agent to treat MI/R injury. cardioprotection; nitrite; left ventricular function; nitrosothiols; reperfusion injury; endothelial nitric oxide synthase

Objectives This paper examined whether nebivolol protects the heart via nitric oxide (NO) synthase and NO-dependent signaling in an in vivo model of acute myocardial infarction. Background Beta3-adrenergic receptor (AR) activation promotes endothelial nitric oxide synthase (eNOS) activity and NO bioavailability. We hypothesized that specific beta3-AR agonists would attenuate myocardial ischemia-reperfusion (MI/R) injury via eNOS activation and increased NO bioavailability. Methods Mice were subjected to 45 min of myocardial ischemia in vivo followed by 24 h of reperfusion (R). Nebivolol (500 ng/kg), CL 316243 (1 μg/kg), BRL-37344 (1 μg/kg), or vehicle (VEH) was administered at the time of R. Myocardial area-at-risk (AAR) and infarct size (INF)/AAR was measured at 24 h of R. Cardiac tissue and plasma were collected to evaluate eNOS phosphorylation, neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase expression, and nitrite and nitrosothiol levels. Results Nebivolol (500 ng/kg) reduced INF/AAR by 37% (p < 0.001 vs. VEH) and serum troponin-I levels from 41 ± 4 ng/ml to 25 ± 4 ng/ml (p < 0.05 vs. VEH). CL 316243 and BRL-37344 reduced INF by 39% and 42%, respectively (p < 0.001 vs. VEH). Nebivolol and CL 316243 increased eNOS phosphorylation at Ser-1177 (p < 0.05 vs. VEH) and increased nitrite and total nitrosylated protein levels. Nebivolol and CL 316243 significantly increased myocardial nNOS expression. Nebivolol failed to reduce INF after MI/R in beta3-AR−/−, eNOS−/−, and in nNOS−/− mice. Conclusions Our results indicate that beta3-AR agonists protect against MI/R injury. Furthermore, the cardioprotective effects of beta3-AR agonists are mediated by rapid eNOS and nNOS activation and increased NO bioavailability.

Many important components of the cardiovascular system display circadian rhythmicity. In both humans and mice, cardiac damage from ischemia/reperfusion (I/R) is greatest at the transition from sleep to activity. The causes of this window of susceptibility are not fully understood. In the murine heart we have reported high amplitude circadian oscillations in expression of the cardioprotective protein regulator of calcineurin 1 (Rcan1). This study was designed to test whether Rcan1 contributes to the circadian rhythm in cardiac protection from I/R damage. Wild type (WT), Rcan1 KO, and Rcan1-Tg mice, with cardiomyocyte-specific overexpression of Rcan1, were subjected to 45 minutes of myocardial ischemia followed by 24 hours of reperfusion. Surgeries were performed either during the first two hours (AM) or the last two hours (PM) of the animal’s light phase. The area at risk was the same for all genotypes at either time point; however, in WT mice, PM-generated infarcts were 78% larger than AM-generated infarcts. Plasma cardiac troponin I levels were likewise greater in PM-operated animals. In Rcan1 KO mice there was no significant difference between the AM- and PM-operated hearts, which displayed greater indices of damage similar to that of PM-operated WT animals. Mice with cardiomyocyte-specific over expression of human RCAN1, likewise, showed no time-of-day difference, but had smaller infarcts comparable to those of AM-operated WT mice. In vitro, cardiomyocytes depleted of RCAN1 were more sensitive to simulated I/R and the calcineurin inhibitor, FK506, restored protection. FK506 also conferred protection to PM-infarcted WT animals. Importantly, transcription of core circadian clock genes was not altered in Rcan1 KO hearts. These studies identify the calcineurin/Rcan1-signaling cascade as a potential therapeutic target through which to benefit from innate circadian changes in cardiac protection without disrupting core circadian oscillations that are essential to cardiovascular, metabolic, and mental health.