The goal of this laboratory exercise is to give upper‐level undergraduate students an introduction to sterile technique in mammalian cell culture and metabolism. The experiment can be completed within a 3‐h lab period and can be performed either in conjunction with other biochemistry/metabolism experiments or used as a stand‐alone experiment. In this experiment, students are tasked with relating the acidification of cell culture medium to metabolism in order to elucidate the mechanism of action for a compound. Students can relate their experimental results to topics covered on glycolysis and oxidative phosphorylation in upper‐level biochemistry classes as well as gain valuable experience relating metabolism to drug discovery.

Our laboratory has previously shown that type 2 diabetic (T2DM) coronary resistance microvessels (CRMs) undergo inward hypertrophic remodeling associated with reduced stiffness. Reduced T2DM CRM tissue stiffness is associated with decreased elastic modulus of coronary vascular smooth muscle cells (VSMCs). The goal of this study was to test the hypothesis that reducing or augmenting coronary vascular cell stiffness will increase or decrease coronary blood flow, respectively. Coronary blood flow was measured from hearts isolated from 16‐week‐old control Db/db and diabetic db/db mice and perfused on a Langendorff system at a constant pressure of 80 mmHg. Latrunculin B (1 μM final concentration; n=2 per group) was infused to depolymerize actin and reduce vascular cell stiffness and jasplakinolide (0.2 μM final concentration; n=4 per group) was infused to increase actin polymerization and increase vascular cell stiffness. After drug infusions, hearts were immediately transferred to an adjacent Langendorff apparatus and were perfusion fixed with 4% paraformaldehyde for 2 mins at 1 mL/min. Hearts were then placed in 4% PFA for 24–48 hours and were stored in 70% EtOH until embedding in paraffin and sectioning. Sections were evaluated for F‐ and G‐actin by immunofluorescence. In pooled Db/db and db/db analyses, Latrunculin B caused an increase in coronary blood flow over baseline (34.1 ± 15.0% above baseline, n=7), and jasplakinolide caused a 26.5 ± 4.1% reduction in coronary blood flow from baseline (n=8). Immunohistochemical analysis confirmed a decreased trend in F/G actin in the coronary microvessels of latrunculin B‐perfused hearts (1.00 vs. 0.82 ± 0.08, n=1–4 per group), while F/G actin tended to be increased in the CRMs of jasplakinolide‐perfused hearts (1.00 ± 0.00 vs. 1.50 ± 0.16, p=0.12, n=2–6 per group). Importantly, the F/G actin ratio in jasplakinolide‐treated hearts was not different measured in the myocardium 50–100 μm away from a CRM (1.00 ± 0.06 in vehicle‐perfused hearts vs. 1.01 ± 0.10 in jasplakinolide‐perfused hearts, p=0.96, n=2–6 per group), demonstrating that the drug was efficacious only in the peri‐vascular region. These data support the hypothesis that coronary blood flow increases as vascular cell stiffness is decreased and coronary blood flow decreases as vascular cell stiffness is increased. Collectively, these data suggest that coronary vascular cell stiffness can modulate coronary blood flow. Support or Funding Information KRM was supported by an American Heart Association Summer Undergraduate Research Fellowship (17UFEL33420025). NIH R00HL116769, R21EB026518, and S10OD023438 to AJT.

The Mediator complex plays an essential role in transcription for nearly all genes, and functions by transmitting physiological and developmental signals to RNA polymerase II. The Mediator complex is regulated by a kinase submodule made up of four proteins: MED12, MED13, CDK8, and Cyclin C. MED12 is a pivotal member of this submodule as it is required for CDK8 activity, which regulates Polymerase II‐driven transcription. The objective of this study is to identify the role of MED12 in the heart and determine how it regulates transcription through interactions with transcription factors. MED12 protein levels are increased in human heart failure biopsies, and in the mouse heart after transverse aortic constriction (TAC)‐induced heart failure. Therefore, we hypothesize that increased levels of MED12 contributes to development of heart failure. To test this hypothesis we generated cardiac‐specific MED12 transgenic (cTg) mice by driving increased MED12 expression in cardiomyocytes using the aMHC promoter. We performed serial echocardiography and observed that increased MED12 expression leads to decreased cardiac function, measured as fractional shortening. Decreased cardiac function was accompanied by increased cardiac chamber size and dilation, without evidence of cardiac fibrosis. We performed RNA sequencing on cTg and control cardiomyocytes, to investigate mechanisms of MED12‐induced cardiac dysfunction, and found that increased MED12 levels lead to dysregulated calcium handing gene expression. MED12 is a transcriptional regulator, but does not directly bind DNA. To determine how MED12 regulates expression of calcium handling genes we used cardiomyocytes from our mouse models to screen for transcription factors that interact with MED12. We immunoprecipitated MED12 and identified interacting proteins by mass spectrometry, many of which are regulators of transcription. In follow‐up experiments we demonstrated that MED12 interacts with the transcription factor (TF) MEF2 to regulate calcium handling genes. Preliminary experiments suggest that MED12 also interacts with TFs CREB and SRF. Additional mechanistic studies are ongoing to determine how MED12 coordinates gene expression through multiple transcription factors in cardiomyocytes. Collectively, our data demonstrate that tight regulation of MED12 levels is necessary to maintain normal cardiac function and regulate gene expression in the heart, and increased MED12 expression leads to the development of heart failure.