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The maintenance of myocardial oxygen supply during stress is essential for sustaining cardiac health. Enhancement of coronary blood flow upon increases in myocardial oxygen demand (i.e., hyperemia) relies on regulation of voltage-gated K+ (Kv) channels by their intracellular β subunits (i.e., Kvβ proteins). Considering that, Kvβ proteins are aldo-keto reductases (AKRs) and regulate Kv channel gating, we tested the hypothesis that elevation of myocardial oxygen demand modifies intracellular NAD(H) in arterial myocytes. Furthermore, we tested whether the resultant change in the redox state of the pyridine nucleotide pool directly regulates coronary Kv1 channel activity. High-resolution imaging mass spectrometry and live-cell fluorescent imaging revealed that augmented cardiac workload significantly increases the cytosolic NADH:NAD+ ratio in intramyocardial arterial myocytes. Intracellular pyridine nucleotide redox ratios reflecting elevated oxygen demand potentiated whole-cell IKv density and stimulated native Kv1 channel activity in a Kvβ2-dependent manner. Mutations in the Kvβ2 catalytic site prevented NADH-induced increases in Kv1 activity, abolished vasodilation in response to elevated L-lactate, and suppressed the relationship between myocardial blood flow and cardiac workload. These results indicate that the AKR activity and pyridine nucleotide sensitivity of Kvβ proteins regulate coronary vasoreactivity and blood flow to the heart during metabolic stress.

The immaturity of engineered cardiac tissues (ECTs) limits their ability to regenerate damaged myocardium and to serve as in vitro disease models and surrogates for drug toxicity testing. Several chronic biomimetic conditioning protocols, including mechanical stretch, perfusion, and electrical stimulation promote ECT maturation but have significant technical limitations. Non-contacting chronic light stimulation using heterologously expressed light-sensitive ChIEF ion channels, termed optogenetics, may be an advantageous alternative to chronic electrical stimulation. As a proof of principle, we transfected ECTs using an AAV packaged ChIEF and then verified acute optical pacing (OP) by patch clamp. We then chronically OP ECTs for 7 days above the intrinsic beat rate. Chronic OP resulted in improved ECT electrophysiological properties; however, ECT force generation and histology remained unchanged. Some changes in cardiac relevant gene expression were noted. This work validates a novel chronic OP paradigm that can be used to identify strategies for optimal in vitro ECT maturation.