PROJECT SUMMARY / ABSTRACT This proposal aims to improve our diagnosis and mechanistic understanding of right ventricular (RV) myocyte dysfunction in heart failure patients with reduced ejection fraction and pulmonary hypertension (HFrEF-PH). Our inability to accurately identify RV failure worsens prognostic and therapeutic efforts in many HFrEF-PH clinical scenarios. RV myocyte contractile reserve indices, such as calcium-activated isometric tension and length-dependent active tension, are reduced in end-stage human HFrEF-PH RV. The latter, a key contributor to chamber Frank-Starling reserve, is severely depressed in HFrEF-PH yet entirely uncaptured by clinical indices. This deficiency highlights one potential reason why clinical tools fall short. A shortcoming of this work was its study of end-stage disease; correlates of myocyte dysfunction from earlier-stage disease would provide more clinically useful insight. Our central hypothesis is that clinical identification of RV myocyte disease in HFrEF-PH requires measuring RV contractile reserve during exercise. Our prior study of primary PH patients supports this, and new data in patients with PH secondary to heart failure do so as well. We also reveal a novel mechanism linking poor recruitment of super-relaxed myosin to poor myocyte length-dependent tension. This links, for the first time in humans, thick filament myosin to RV myocyte and chamber reserve. Dissecting these findings will clarify mechanism and help guide the use of novel direct-acting sarcomere drugs for such myocyte deficits. We will test our hypothesis via three Specific Aims. In Aim 1, we test whether RV exercise reserve measures are better than resting RV measures at identifying RV myocyte contractile dysfunction. We will do this by prospectively measuring rest and exercise RV function using clinical and pressure-volume loop-derived RV indices and comparing their ability to identify intrinsic RV myocyte contractile failure, the latter ascertained from concurrently obtained RV biopsies. In Aim 2, we test whether hypophosphorylation of protein kinase A- protein targets or hyperphosphorylation of sarcomere Z-disc scaffolding proteins blunts length-dependent activation in HFrEF-PH RV myocytes. We will assess myocytes under the influence of select kinases and phosphatases, assess thick filament super-relaxed myosin recruitment, and test the effect of specific modifications on length-dependent force in human engineered heart tissue. In Aim 3, we test the ability of novel direct-acting sarcomere drugs, termed myotropes, on ex vivo HFrEF RV myocyte length-dependent tension. We also test whether newfound Aim 1 clinical measures identify patients with better drug response. Through this proposal, we expect to deliver novel clinical indicators of intrinsic HFrEF-PH RV myocyte failure. By coupling these clinical indicators to biophysical mechanisms and drug responses, we hope to usher in novel treatments for RV failure. These goals, which al...