Blockade of PI3Kγ may provide a dual therapeutic advantage in cancer therapy by simultaneously preventing anthracyclines cardiotoxicity and reducing tumor growth.

Cardiomyocyte proteostasis is mediated by the ubiquitin/proteasome system (UPS) and autophagy/lysosome system and is fundamental for cardiac adaptation to both physiologic (e.g., exercise) and pathologic (e.g., pressure overload) stresses. Both the UPS and autophagy/lysosome system exhibit reduced efficiency as a consequence of aging, and dysfunction in these systems is associated with cardiomyopathies. The musclespecific ubiquitin ligase atrogin-1 targets signaling proteins involved in cardiac hypertrophy for degradation. Here, using atrogin-1 KO mice in combination with in vivo pulsed stable isotope labeling of amino acids in cell culture proteomics and biochemical and cellular analyses, we identified charged multivesicular body protein 2B (CHMP2B), which is part of an endosomal sorting complex (ESCRT) required for autophagy, as a target of atrogin-1-mediated degradation. Mice lacking atrogin-1 failed to degrade CHMP2B, resulting in autophagy impairment, intracellular protein aggregate accumulation, unfolded protein response activation, and subsequent cardiomyocyte apoptosis, all of which increased progressively with age. Cellular proteostasis alterations resulted in cardiomyopathy characterized by myocardial remodeling with interstitial fibrosis, with reduced diastolic function and arrhythmias. CHMP2B downregulation in atrogin-1 KO mice restored autophagy and decreased proteotoxicity, thereby preventing cell death. These data indicate that atrogin-1 promotes cardiomyocyte health through mediating the interplay between UPS and autophagy/lysosome system and its alteration promotes development of cardiomyopathies.

Cardiac sympathetic neurons (SNs) finely tune the rate and strength of heart contractions to match blood demand, both at rest and during acute stress, through the release of noradrenaline (NE). Junctional sites at the interface between the two cell types have been observed, although whether direct neurocardiac coupling has a role in heart physiology has not been clearly demonstrated to date. We investigated the dynamics of SN/cardiomyocyte intercellular signalling, both by fluorescence resonance energy transfer-based imaging of cAMP in co-cultures, as a readout of cardiac β-adrenergic receptor activation, and in vivo, using optogenetics in transgenic mice with SN-specific expression of Channelrhodopsin-2. We demonstrate that SNs and cardiomyocytes interact at specific sites in the human and rodent heart, as well as in co-cultures. Accordingly, neuronal activation elicited intracellular cAMP increases only in directly contacted myocytes and cell-cell coupling utilized a junctional extracellular signalling domain with an elevated NE concentration. In the living mouse, optogenetic activation of cardiac SNs innervating the sino-atrial node resulted in an instantaneous chronotropic effect, which shortened the heartbeat interval with single beat precision. Remarkably, inhibition of the optogenetically elicited chronotropic responses required a high dose of propranolol (20-50 mg kg ), suggesting that sympathetic neurotransmission in the heart occurs at a locally elevated NE concentration. Our in vitro and in vivo data suggest that the control of cardiac function by SNs occurs via direct intercellular coupling as a result of the establishment of a specific junctional site.

Extrasystoles lead to several consequences, ranging from uneventful palpitations to lethal ventricular arrhythmias, in the presence of pathologies, such as myocardial ischemia. The role of working versus conducting cardiomyocytes, as well as the tissue requirements (minimal cell number) for the generation of extrasystoles, and the properties leading ectopies to become arrhythmia triggers (topology), in the normal and diseased heart, have not been determined directly in vivo. Here, we used optogenetics in transgenic mice expressing ChannelRhodopsin-2 selectively in either cardiomyocytes or the conduction system to achieve cell typespecific, noninvasive control of heart activity with high spatial and temporal resolution. By combining measurement of optogenetic tissue activation in vivo and epicardial voltage mapping in Langendorffperfused hearts, we demonstrated that focal ectopies require, in the normal mouse heart, the simultaneous depolarization of at least 1,300-1,800 working cardiomyocytes or 90-160 Purkinje fibers. The optogenetic assay identified specific areas in the heart that were highly susceptible to forming extrasystolic foci, and such properties were correlated to the local organization of the Purkinje fiber network, which was imaged in three dimensions using optical projection tomography. Interestingly, during the acute phase of myocardial ischemia, focal ectopies arising from this location, and including both Purkinje fibers and the surrounding working cardiomyocytes, have the highest propensity to trigger sustained arrhythmias. In conclusion, we used cell-specific optogenetics to determine with high spatial resolution and cell type specificity the requirements for the generation of extrasystoles and the factors causing ectopies to be arrhythmia triggers during myocardial ischemia.optogenetics | heart | Purkinje fiber | arrhythmia | cardiac ectopies A berrant heartbeats, caused by the ectopic depolarization of a group of cardiomyocytes, are associated with a wide range of consequences, from the commonly experienced feeling of "palpitation" to the triggering of potentially lethal ventricular arrhythmias in diseased hearts. Physiological conduction of normal heartbeats is orchestrated by the interaction of at least two functionally and anatomically distinct populations of cardiomyocytes: the working cardiomyocytes and the conduction system (i.e., Purkinje fibers at the ventricular level) (1). The electrotonic coupling of myocardial cells protects the heart from abnormal excitation and allows the effect of spontaneous activity in sparse cardiomyocytes to be "sunk" by the surrounding myocardium. As a result, a minimal "critical" number of cardiomyocytes needs to simultaneously depolarize to prevail over such a protective mechanism and generate conducted beats (2-5). When this occurs, the source-sink mismatch (abnormal depolarization current/ myocardial electrotonic sink) is focally overcome, resulting in a premature ventricular contraction (PVC) that, in the presence of arrhythmogenic substrates, may e...

Cardiac SNs are strong regulators of the cardiomyocyte size via β2-AR-dependent repression of proteolysis, demonstrating that the neuro-cardiac axis operates constitutively for the determination of the physiological cardiomyocyte size. These results are of great clinical relevance given the role of β-AR in cardiovascular diseases and their modulation in therapy.

Key points The heart is innervated by a dense sympathetic neuron network which, in the short term, controls chronotropy and inotropy and, in the long term, regulates cardiomyocyte size. Acute neurogenic control of heart rate is achieved locally through direct neuro‐cardiac coupling at specific junctional sites (neuro‐cardiac junctions). The ventricular sympathetic network topology is well‐defined and characteristic for each mammalian species. In the present study, we used cell size regulation to determine whether long‐term modulation of cardiac structure is achieved via direct sympatho‐cardiac coupling. Local density of cardiac innervation correlated with cell size throughout the myocardial walls in all mammalian species analysed, including humans. The data obtained suggest that constitutive neurogenic control of cardiomyocyte trophism occurs through direct intercellular signalling at neuro‐cardiac junctions. Abstract It is widely appreciated that sympathetic stimulation of the heart involves a sharp increase in beating rate and significant enhancement of contractility. We have previously shown that, in addition to these evident functions, sympathetic neurons (SNs) also provide trophic input to cardiomyocytes (CMs), regulating cell and organ size. More recently, we have demonstrated that cardiac neurons establish direct interactions with CMs, allowing neuro‐cardiac communication to occur locally, with a ‘quasi‐synaptic’ mechanism. Based on the evidence that cardiac SNs are unevenly distributed throughout the myocardial walls, we investigated the hypothesis that CM size distribution reflects the topology of neuronal density. In vitro analyses of SN/CM co‐cultures, ex vivo confocal and multiphoton imaging in clarified hearts, and biochemical and molecular approaches were employed, in both rodent and human heart biopsies. In line with the trophic effect of SNs, and with local neuro‐cardiac communication, CMs, directly contacted by SNs in co‐cultures, were larger than the non‐targeted ones. This property reflects the distribution of CM size throughout the ventricles of intact mouse heart, in which cells in the outer myocardial layers, which were contacted by more neuronal processes, were larger than those in the less innervated subendocardial region. Such differences disappeared upon genetic or pharmacological interference with the trophic SN/CM signalling axis. Remarkably, CM size followed the SN distribution pattern in other mammals, including humans. Our data suggest that both the acute and chronic influence of SNs on cardiac function and structure is enacted as a result of the establishment of specific intercellular neuro‐cardiac junctions.

SummarySo far, opposing outcomes have been reported following neonatal apex resection in mice, questioning the validity of this injury model to investigate regenerative mechanisms. We performed a systematic evaluation, up to 180 days after surgery, of the pathophysiological events activated upon apex resection. In response to cardiac injury, we observed increased cardiomyocyte proliferation in remote and apex regions, neovascularization, and local fibrosis. In adulthood, resected hearts remain consistently shorter and display permanent fibrotic tissue deposition in the center of the resection plane, indicating limited apex regrowth. However, thickening of the left ventricle wall, explained by an upsurge in cardiomyocyte proliferation during the initial response to injury, compensated cardiomyocyte loss and supported normal systolic function. Thus, apex resection triggers both regenerative and reparative mechanisms, endorsing this injury model for studies aimed at promoting cardiomyocyte proliferation and/or downplaying fibrosis.

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