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During myocardial ischemia, upregulation of the hedgehog (Hh) pathway promotes neovascularization and increases cardiomyocyte survival. The canonical Hh pathway activates a transcriptional program through the Gli family of transcription factors by derepression of the seven-transmembrane protein smoothened (Smo). The mechanisms linking Smo to Gli are complex and, in some cell types, involve coupling of Smo to G¡ proteins. In the present study, we investigated, for the first time, the transcriptional response of cardiomyocytes to sonic hedgehog (Shh) and the role of G¡ protein utilization. Our results show that Shh strongly activates Gli1 expression by quantitative PCR in a Smodependent manner in neonatal rat ventricular cardiomyocytes. Microarray analysis of gene expression changes elicited by Shh and sensitive to a Smo inhibitor identified a small subset of 37 cardiomyocyte- specific genes regulated by Shh, including some in the PKA and purinergic signaling pathways. In addition, neonatal rat ventricular cardiomyocytes infected with an adenovirus encoding GiCT, a peptide that impairs receptor-Gi protein coupling, showed reduced activation of Hh targets. In vitro data were confirmed in transgenic mice with cardiomyocyte-inducible GiCT expression. Transgenic GiCT mice showed specific reduction of Gli1 expression in the heart under basal conditions and failed to upregulate the Hh pathway upon ischemia and reperfusion injury, unlike their littermate controls. This study characterizes, for the first time, the transcriptional response of cardiomyocytes to Shh and establishes a critical role for Smo coupling to G¡ in Hh signaling in the normal and ischemic myocardium. [ABSTRACT FROM AUTHOR]

Simple Summary: Two Israeli studies about medical marijuana potentially interfering with immunotherapies like nivolumab for cancer treatment have received substantial attention. However, there have been anonymous but detailed concerns about these reports on PubPeer. This team attempted to verify the data analysis and statistics of these two reports and the published correction. Many findings, including some that could impact the statistical conclusions, could not be verified. Of 22 statistical in the prospective report, 4 could not be repeated using the same statistics or with the provided N. The p-value on 17 corresponded with that of a different statistical test than was listed in the methods. Re-analysis also identified some previously unreported significant differences (e.g., age) between cannabis users and non-users at baseline. Further study of the safety of immunotherapy and cannabis combination may be warranted using patient groups that have been matched on key demographic and medical variables. A retrospective (N = 140) and a prospective (N = 102) observational Israeli study by Bar-Sela and colleagues about cannabis potentially adversely impacting the response to immunotherapy have together been cited 202 times, including by clinical practice guidelines. There have also been concerns on PubPeer outlining irregularities and unverifiable information in their statistics and numerous errors in calculating percentages. This reanalysis attempted to verify the data analysis while including non-parametric statistics. The corrected prospective report contained 22 p-values, but only one (4.5%) could be verified despite the authors being transparent about the N and statistics employed. Cannabis users were significantly (p < 0.0025) younger than non-users, but this was not reported in the retrospective report. There were also errors in percentage calculations (e.g., 13/34 reported as 22.0% instead of 38.2%). Overall, these observational investigations, and especially the prospective, appear to contain gross inaccuracies which could impact the statistical decisions (i.e., significant findings reported as non-significant or vice-versa). Although it is mechanistically plausible that cannabis could have immunosuppressive effects which inhibit the response to immunotherapy, these two reports should be viewed cautiously. Larger prospective studies of this purported drug interaction that account for potential confounds (e.g., greater nicotine smoking among cannabis users) may be warranted. [ABSTRACT FROM AUTHOR]