Cardiopoietic programming of embryonic stem cells for tumor-free heart repair

Embryonic stem cells have the distinct potential for tissue regeneration, including cardiac repair. Their propensity for multilineage differentiation carries, however, the liability of neoplastic growth, impeding therapeutic application. Here, the tumorigenic threat associated with embryonic stem cell transplantation was suppressed by cardiac-restricted transgenic expression of the reprogramming cytokine TNF-α, enhancing the cardiogenic competence of recipient heart. The in vivo aptitude of TNF-α to promote cardiac differentiation was recapitulated in embryoid bodies in vitro. The procardiogenic action required an intact endoderm and was mediated by secreted cardio-inductive signals. Resolved TNF-α–induced endoderm-derived factors, combined in a cocktail, secured guided differentiation of embryonic stem cells in monolayers produce cardiac progenitors termed cardiopoietic cells. Characterized by a down-regulation of oncogenic markers, up-regulation, and nuclear translocation of cardiac transcription factors, this predetermined population yielded functional cardiomyocyte progeny. Recruited cardiopoietic cells delivered in infarcted hearts generated cardiomyocytes that proliferated into scar tissue, integrating with host myocardium for tumor-free repair. Thus, cardiopoietic programming establishes a strategy to hone stem cell pluripotency, offering a tumor-resistant approach for regeneration

Myokine Musclin Is Critical for Exercise-Induced Cardiac Conditioning

This study investigates the role and mechanisms by which the myokine musclin promotes exercise-induced cardiac conditioning. Exercise is one of the most powerful triggers of cardiac conditioning with proven benefits for healthy and diseased hearts. There is an emerging understanding that muscles produce and secrete myokines, which mediate local and systemic “crosstalk” to promote exercise tolerance and overall health, including cardiac conditioning. The myokine musclin, highly conserved across animal species, has been shown to be upregulated in response to physical activity. However, musclin effects on exercise-induced cardiac conditioning are not established. Following completion of a treadmill exercise protocol, wild type (WT) mice and mice with disruption of the musclin-encoding gene, Ostn, had their hearts extracted and exposed to an ex vivo ischemia-reperfusion protocol or biochemical studies. Disruption of musclin signaling abolished the ability of exercise to mitigate cardiac ischemic injury. This impaired cardioprotection was associated with reduced mitochondrial content and function linked to blunted cyclic guanosine monophosphate (cGMP) signaling. Genetic deletion of musclin reduced the nuclear abundance of protein kinase G (PKGI) and cyclic adenosine monophosphate (cAMP) response element binding (CREB), resulting in suppression of the master regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), and its downstream targets in response to physical activity. Synthetic musclin peptide pharmacokinetic parameters were defined and used to calculate the infusion rate necessary to maintain its plasma level comparable to that observed after exercise. This infusion was found to reproduce the cardioprotective benefits of exercise in sedentary WT and Ostn-KO mice. Musclin is essential for exercise-induced cardiac protection. Boosting musclin signaling might serve as a novel therapeutic strategy for cardioprotection

Loss of ATP-Sensitive Potassium Channel Surface Expression in Heart Failure Underlies Dysregulation of Action Potential Duration and Myocardial Vulnerability to Injury.

The search for new approaches to treatment and prevention of heart failure is a major challenge in medicine. The adenosine triphosphate-sensitive potassium (KATP) channel has been long associated with the ability to preserve myocardial function and viability under stress. High surface expression of membrane KATP channels ensures a rapid energy-sparing reduction in action potential duration (APD) in response to metabolic challenges, while cellular signaling that reduces surface KATP channel expression blunts APD shortening, thus sacrificing energetic efficiency in exchange for greater cellular calcium entry and increased contractile force. In healthy hearts, calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the Kir6.2 KATP channel subunit initiating a cascade responsible for KATP channel endocytosis. Here, activation of CaMKII in a transaortic banding (TAB) model of heart failure is coupled with a 35-40% reduction in surface expression of KATP channels compared to hearts from sham-operated mice. Linkage between KATP channel expression and CaMKII is verified in isolated cardiomyocytes in which activation of CaMKII results in downregulation of KATP channel current. Accordingly, shortening of monophasic APD is slowed in response to hypoxia or heart rate acceleration in failing compared to non-failing hearts, a phenomenon previously shown to result in significant increases in oxygen consumption. Even in the absence of coronary artery disease, failing myocardium can be further injured by ischemia due to a mismatch between metabolic supply and demand. Ischemia-reperfusion injury, following ischemic preconditioning, is diminished in hearts with CaMKII inhibition compared to wild-type hearts and this advantage is largely eliminated when myocardial KATP channel expression is absent, supporting that the myocardial protective benefit of CaMKII inhibition in heart failure may be substantially mediated by KATP channels. Recognition of CaMKII-dependent downregulation of KATP channel expression as a mechanism for vulnerability to injury in failing hearts points to strategies targeting this interaction for potential preventives or treatments

Musclin, A Myokine Induced by Aerobic Exercise, Retards Muscle Atrophy During Cancer Cachexia in Mice

Physical activity improves the prognosis of cancer patients, partly by contrasting the associated muscle wasting (cachexia), through still unknown mechanisms. We asked whether aerobic exercise causes secretion by skeletal muscles of proteins (myokines) that may contrast cachexia. Media conditioned by peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α)-expressing myotubes, reproducing some metabolic adaptations of aerobic exercise, as increased mitochondrial biogenesis and oxidative phosphorylation, restrained constitutively active Forkhead box-containing subfamily O3 (caFoxO3)-induced proteolysis. Microarray analysis identified amphiregulin (AREG), natriuretic peptide precursor B (NppB), musclin and fibroblast growth factor 18 (FGF18) as myokines highly induced by PGC1α. Notably, only musclin tended to be low in muscle of mice with a rare human renal carcinoma; it was reduced in plasma and in muscles of C26-bearing mice and in atrophying myotubes, where PGC1α expression is impaired. Therefore, we electroporated the Tibialis Anterior (TA) of C26-bearing mice with musclin or (its receptor) natriuretic peptide receptor 3 (Npr3)-encoding plasmids and found a preserved fiber area, as a result of restrained proteolysis. Musclin knockout (KO) mice lose more muscle tissue during growth of two distinct cachexia-causing tumors. Running protected C26-bearing mice from cachexia, not changing tumor growth, and rescued the C26-induced downregulation of musclin in muscles and plasma. Musclin expression did not change in overloaded plantaris of mice, recapitulating partially muscle adaptations to anaerobic exercise. Musclin might, therefore, be beneficial to cancer patients who cannot exercise and are at risk of cachexia and may help to explain how aerobic exercise alleviates cancer-induced muscle wasting

Monophasic action potential duration shortening is blunted in failing hearts.

<p>Monophasic action potentials were measured from a single left ventricular epicardial position in isolated hearts from TAB and sham mice before and during exposure to hypoxia or abrupt heart rate acceleration from 400 beats per minute (bpm) to 667 bpm (150 msec cycle length to 90 msec cycle length) driven by pacing. <b>A)</b> Representative normalized monophasic action potentials at baseline (solid lines) and in response to hypoxia (dotted lines) from hearts of sham (left panel) and TAB (right panel) mice. A horizontal line designates the point at which monophasic action potential duration at 90% repolarization (MAPD₉₀) was measured. <b>B)</b> Summary of MAPD₉₀ shortening (left), half-time (t_1/2) of MAPD₉₀ shortening (middle), and rate of MAPD₉₀ shortening (right) in hearts from sham and TAB mice in response to hypoxia (*p < .05). <b>C)</b> Summary of MAPD₉₀ shortening (left), half-time (t_1/2) of MAPD₉₀ shortening (middle), and rate of MAPD₉₀ shortening (right) in hearts from sham and TAB mice in response to heart rate acceleration (*p < .05).</p

Dynamic downregulation of K_ATP channel current density is blunted in ventricular cardiomyocytes of TAB mice.

<p><b>A)</b> Representative tracings of pinacidil (PIN, 100 μM)- and 2,4-dinitrophenol (DNP, 200 μM)-stimulated K_ATP channel current density in response to isoproterenol (ISO, 1 μM). <b>B)</b> Summary of percent inhibition of pinacidil and DNP-stimulated K_ATP channel current density by isoproterenol (1 μM) in isolated cardiomyocytes of sham and TAB mice (**p<0.01 <i>vs</i>. sham).</p

K_ATP channel surface expression is down-regulated in ventricles of hearts after transverse aortic banding.

<p>A) Representative western blots of the K_ATP Kir6.2 subunit and the sodium-potassium pump (Na⁺-K⁺ pump) from the biotinylated membrane fraction of ventricular tissue from isolated hearts of sham and TAB mice. <b>B)</b> Summary of ventricular membrane Kir6.2 expression normalized to Na⁺-K⁺ pump expression in hearts of sham and TAB mice (*p<0.05 <i>vs</i>. sham). <b>C)</b> Representative current profiles of isolated left ventricular myocytes before and after application of the K_ATP channel activators, pinacidil (PIN, 100 μM) and 2,4-dinitrophenol (DNP, 200 μM). <b>D)</b> Summary of K_ATP channel current density (after-before K_ATP channel activations) from isolated ventricular cardiomyocytes of sham and TAB mice (**p<0.01 <i>vs</i>. sham).</p

Morphological and electrophysiological changes in transverse aortic banding (TAB) <i>vs</i>. sham operated mice.

<p><b>A)</b> Representative parasternal long axis echocardiographic images of the ventricles of sham and TAB operated mice. <b>B)</b> Summary data of left ventricular ejection fraction (**p<0.01 <i>vs</i>. sham). <b>C)</b> Representative action potentials recorded in isolated ventricular cardiomyocytes from sham and TAB mice. <b>D)</b> Summary data of action potential duration in isolated ventricular myocytes of sham and TAB mice (**p<0.01 <i>vs</i>. sham). APD₅₀, APD₇₅, APD₉₀: action potential duration at 50%, 75% and 90% repolarization, respectively. E_m: membrane potential.</p