Abstract Body: The Ras-Raf-MEK-ERK MAP kinase pathway (MEK/ERK signaling) plays a pivotal role in many cell types, including those in the heart. Hyperactivation of MEK/ERK signaling contributes to oncogenesis in about 30% of cancers, prompting the development of multiple inhibitors of this pathway for the treatment of cancer. MEK inhibitors (MEKi), most commonly used for the treatment of BRAF V600E-mutated melanoma, have been shown to induce cardiotoxicity, leading to a renewed interest in the role of this pathway in the heart. Recently, our laboratory demonstrated that Trametinib (Trm), a highly specific FDA-approved MEKi, induces reversible heart failure in adult mice, mimicking what is observed in some human patients. Trm caused mitochondrial dysfunction and induced an innate immune response in the hearts of these mice. These findings would not have been expected based on what is currently understood about MEK/ERK signaling in the heart. To test whether these cardiotoxic side effects of Trm and other MEKi’s arise from on-target inhibition of MEK/ERK signaling in the heart or from an off-target effect, we generated a novel cardiomyocyte-specific MEK1 knockout (CM-MEK1KO) mouse model. CM-MEK1KO mice have significantly reduced contractile function compared to WT mice as early as 5 weeks of age. Serial echocardiograms demonstrated that contractile function worsens over time and CM-MEK1KO mice die prematurely, beginning at 4 months of age. Though previous studies have suggested that MEK/ERK signaling is pro-hypertrophic in the injured mouse heart, CM-MEK1KO mice actually had higher heart weights and cardiomyocyte cross sectional area compared to wild type littermates. Bulk RNA sequencing demonstrated broad transcriptional changes in the hearts of CM-MEK1KO mice. Pathways involved in cardiac hypertrophy and the response to injury were overexpressed while some genes associated with mitochondria decreased in abundance. Collectively these data indicate that the MEK1-ERK signaling axis is critical for normal cardiac function and at least some of the cardiotoxic side effects of MEKi’s likely arise through inhibition of MEK/ERK signaling in cardiomyocytes. Ongoing experiments are testing whether CM-MEK1KO mice exhibit mitochondrial dysfunction, possibly substantiating a novel physiological role for the MEK/ERK signaling pathway in the heart.