SUMMARY Cardiac contractility is mediated by variable flux in intracellular calcium (Ca2+), thought to be integrated into mitochondria via the mitochondrial calcium uniporter (MCU) channel to match energetic demand. Here we examine a conditional, cardiomyocyte-specific, mutant mouse lacking Mcu, the pore-forming subunit of the MCU channel, in adulthood. Mcu−/− mice display no overt baseline phenotype and are protected against mCa2+-overload in an in vivo myocardial ischemia-reperfusion injury model by preventing the activation of the mitochondrial permeability transition pore, decreasing infarct size, and preserving cardiac function. In addition, we find that Mcu−/− mice lack contractile responsiveness to acute β-adrenergic receptor stimulation and in parallel are unable to activate mitochondrial dehydrogenases and display reduced bioenergetic reserve capacity. These results support the hypothesis that MCU may be dispensable for homeostatic cardiac function but required to modulate Ca2+-dependent metabolism during acute stress.

Mitochondrial calcium ( m Ca 2+ ) has a central role in both metabolic regulation and cell death signalling, however its role in homeostatic function and disease is controversial 1 . Slc8b1 encodes the mitochondrial Na + /Ca 2+ exchanger (NCLX), which is proposed to be the primary mechanism for m Ca 2+ extrusion in excitable cells 2,3 . Here we show that tamoxifen-induced deletion of Slc8b1Reprints and permissions information is available at www.nature.com/reprints.

Rationale GDF11 (Growth Differentiation Factor 11) is a member of the transforming growth factor β (TGFβ) super family of secreted factors. A recent study showed that reduced GDF11 blood levels with aging was associated with pathological cardiac hypertrophy (PCH), and restoring GDF11 to normal levels in old mice rescued PCH. Objective To determine if and by what mechanism GDF11 rescues aging dependent PCH. Methods and Results 24-month-old C57BL/6 mice were given a daily injection of either recombinant (r) GDF11 at 0.1mg/kg or vehicle for 28 days. rGDF11 bioactivity was confirmed in-vitro. After treatment, rGDF11 levels were significantly increased but there was no significant effect on either heart weight (HW) or body weight (BW). HW/BW ratios of old mice were not different from 8 or 12 week-old animals, and the PCH marker ANP was not different in young versus old mice. Ejection fraction, internal ventricular dimension, and septal wall thickness were not significantly different between rGDF11 and vehicle treated animals at baseline and remained unchanged at 1, 2 and 4 weeks of treatment. There was no difference in myocyte cross-sectional area rGDF11 versus vehicle-treated old animals. In vitro studies using phenylephrine-treated neonatal rat ventricular myocytes (NRVM), to explore the putative anti-hypertrophic effects of GDF11, showed that GDF11 did not reduce NRVM hypertrophy, but instead induced hypertrophy. Conclusions Our studies show that there is no age-related PCH in disease free 24-month-old C57BL/6 mice and that restoring GDF11 in old mice has no effect on cardiac structure or function.

Aims: Mitochondrial Ca 2+ homeostasis is crucial for balancing cell survival and death. The recent discovery of the molecular identity of the mitochondrial Ca 2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca 2+ regulation in various cell types, including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca 2+ entry through posttranslational modifications of MCU are completely unknown. Therefore, we investigated a 1 -adrenergic-mediated signal transduction of MCU posttranslational modification and function in cardiac cells. Results: a 1 -adrenoceptor (a 1 -AR) signaling translocated activated proline-rich tyrosine kinase 2 (Pyk2) from the cytosol to mitochondrial matrix and accelerates mitochondrial Ca 2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, we found that a 1 -AR stimulation increases reactive oxygen species production at mitochondria, mitochondrial permeability transition pore activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca 2+ overload. Innovation: Our data indicate that inhibition of a 1 -AR-Pyk2-MCU signaling represents a potential novel therapeutic target to limit or prevent mitochondrial Ca 2+ overload, oxidative stress, mitochondrial injury, and myocardial death during pathophysiological conditions, where chronic adrenergic stimulation is present. Conclusion: The a 1 -AR-Pyk2-dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca 2+ entry and apoptosis in cardiac cells. Antioxid. Redox Signal. 21, 863-879.

Rationale Catecholamines increase cardiac contractility, but exposure to high concentrations or prolonged exposures can cause cardiac injury. A recent study demonstrated that a single subcutaneous injection of isoproterenol (ISO; 200 mg/kg) in mice causes acute myocyte death (8-10%) with complete cardiac repair within a month. Cardiac regeneration was via endogenous cKit+ cardiac stem cell (CSC)-mediated new myocyte formation. Objective Our goal was to validate this simple injury/regeneration system and use it to study the biology of newly forming adult cardiac myocytes. Methods and Results C57BL/6 mice (n=173) were treated with single injections of vehicle, 200mg/kg or 300mg/kg ISO, or with two daily doses of 200mg/kg ISO for 6 days. Echocardiography revealed transiently increased systolic function and unaltered diastolic function 1 day after single ISO injection. Single ISO injections also caused membrane injury in about 10% of myocytes but few of these myocytes appeared to be necrotic. Circulating troponin I levels after ISO were elevated, further documenting myocyte damage. However, myocyte apoptosis was not increased after ISO injury. Heart weight to body weight ratio and fibrosis were also not altered 28 days after ISO injection. Single or multiple dose ISO injury was not associated with an increase in the percentage of 5-ethynyl-2’-deoxyuridine (EdU)-labeled myocytes. Furthermore, ISO injections did not increase new myocytes in cKit+/Cre × R-GFP transgenic mice. Conclusions A single dose of ISO causes injury in about 10% of the cardiomyocytes. However, most of these myocytes appear to recover and do not elicit cKit+ cardiac stem cell (CSC)-derived myocyte regeneration.

ϩ influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca 2ϩ influx is mainly mediated by the mitochondrial Ca 2ϩ uniporter (MCU). Growing evidence also indicates that mitochondrial Ca 2ϩ influx mechanisms are regulated not solely by MCU but also by multiple channels/transporters. We have previously reported that skeletal muscle-type ryanodine receptor (RyR) type 1 (RyR1), which expressed at the mitochondrial inner membrane, serves as an additional Ca 2ϩ uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca 2ϩ influx mechanism is the dominant regulator of mitochondrial morphology/dynamics and energetics in cardiomyocytes. To investigate the role of mitochondrial RyR1 in the regulation of mitochondrial morphology/function in cardiac cells, RyR1 was transiently or stably overexpressed in cardiac H9c2 myoblasts. We found that overexpressed RyR1 was partially localized in mitochondria as observed using both immunoblots of mitochondrial fractionation and confocal microscopy, whereas RyR2, the main RyR isoform in the cardiac sarcoplasmic reticulum, did not show any expression at mitochondria. Interestingly, overexpression of RyR1 but not MCU or RyR2 resulted in mitochondrial fragmentation. These fragmented mitochondria showed bigger and sustained mitochondrial Ca 2ϩ transients compared with basal tubular mitochondria. In addition, RyR1-overexpressing cells had a higher mitochondrial ATP concentration under basal conditions and showed more ATP production in response to cytosolic Ca 2ϩ elevation compared with nontransfected cells as observed by a matrix-targeted ATP biosensor. These results indicate that RyR1 possesses a mitochondrial targeting/retention signal and modulates mitochondrial morphology and Ca 2ϩ -induced ATP production in cardiac H9c2 myoblasts. fluorescence resonance energy transfer; mitochondrial Ca 2ϩ uniporter; mitochondria; mitochondrial morphology; ryanodine receptor type 1 MITOCHONDRIAL Ca 2ϩ is critical for the regulation of various cellular functions, including energy metabolism, ROS generation, spatiotemporal dynamics of Ca 2ϩ signaling, and cell growth/development and death (11,18,23). Historically, Ca 2ϩ was found to be accumulated by mitochondria over 5 decades ago (for reviews, see Refs. 24, 64, and 69), and, shortly thereafter, it was also recognized that Ca 2ϩ stimulates the oxidative phosphorylation [tricarboxylic acid (TCA) cycle] and electron transport chain activity, which results in the stimulation of ATP synthesis (for a review, see Ref. 23). Additionally, the coexistence of mitochondrial dysfunction and loss of cellular Ca 2ϩ homeostasis are frequently observed in various cardiovascular diseases, but it is still not clear how altered mitochondrial Ca 2ϩ handing and/or mitochondrial dysfunction are involved in the pathogenesis of each different disease setting (23,33,56).Although the basic functional and pharmacological properties of mitochondrial ...

Rationale Cortical bone stem cells (CBSCs) have been shown to reduce ventricular remodeling and improve cardiac function in a murine myocardial infarction (MI) model. These effects were superior to other stem cell types that have been used in recent early stage clinical trials. However, CBSC efficacy has not been tested in a preclinical large animal model using approaches that could be applied to patients. Objective To determine if post–MI transendocardial injection of allogeneic CBSCs reduces pathological structural and functional remodeling and prevents the development of heart failure in a swine MI model. Methods and Results Female Göttingen swine underwent left anterior descending coronary artery occlusion, followed by reperfusion (ischemia–reperfusion MI). Animals received, in a randomized, blinded manner, 1:1 ratio, CBSCs (n = 9) (2×107 cells total) or placebo (vehicle; VEH, n = 9) through NOGA® guided transendocardial injections. 5–ethynyl–2’deoxyuridine (EdU), a thymidine analog, containing minipumps were inserted at the time of MI induction. At 72hrs (n=8) initial injury and cell retention were assessed. At 3 Months post–MI, cardiac structure and function was evaluated by serial echocardiography, and terminal invasive hemodynamics. CBSCs were present in the MI border zone and proliferating at 72hrs post–MI but had no effect on initial cardiac injury or structure. At 3 months, CBSC–treated hearts had significantly reduced scar size, smaller myocytes and increased myocyte nuclear density. Noninvasive echocardiographic measurements showed that left ventricular (LV) volumes and ejection fraction were significantly more preserved in CBSC–treated hearts and invasive hemodynamic measurements documented improved cardiac structure and functional reserve. The number of EdU+ cardiac myocytes was increased in CBSC– vs. VEH– treated animals. Conclusions CBSC administration into the MI border zone reduces pathological cardiac structural and functional remodeling and improves LV functional reserve. These effects reduce those processes that can lead to heart failure with reduced ejection fraction (HFrEF).

Rationale Sorafenib is an effective treatment for renal cell carcinoma, but recent clinical reports have documented its cardiotoxicity through an unknown mechanism. Objective Determining the mechanism of sorafenib-mediated cardiotoxicity. Methods and Results Mice treated with sorafenib or vehicle for 3 weeks underwent induced myocardial infarction (MI) after 1 week of treatment. Sorafenib markedly decreased 2-week survival relative to vehicle-treated controls but echocardiography at 1 and 2 weeks post-MI detected no differences in cardiac function. Sorafenib-treated hearts had significantly smaller diastolic and systolic volumes and reduced heart weights. High doses of sorafenib induced necrotic death of isolated myocytes in vitro, but lower doses did not induce myocyte death or affect inotropy. Histological analysis documented increased myocyte cross-sectional area despite smaller heart sizes following sorafenib treatment, further suggesting myocyte loss. Sorafenib caused apoptotic cell death of cardiac- and bone-derived c-kit+ stem cells in vitro and decreased the number of BrdU+ myocytes detected at the infarct border zone in fixed tissues. Sorafenib had no effect on infarct size, fibrosis or post-MI neovascularization. When sorafenib-treated animals received metoprolol treatment post-MI, the sorafenib-induced increase in post MI mortality was eliminated, cardiac function was improved, and myocyte loss was ameliorated. Conclusions Sorafenib cardiotoxicity results from myocyte necrosis rather than from any direct effect on myocyte function. Surviving myocytes undergo pathological hypertrophy. Inhibition of c-kit+ stem cell proliferation by inducing apoptosis exacerbates damage by decreasing endogenous cardiac repair. In the setting of MI, which also causes large-scale cell loss, sorafenib cardiotoxicity dramatically increases mortality.