Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome effecting more than 3 million people in the United States alone. HFpEF patients have a 5-year survival rate of less than 50% with no change in prognosis over the last 30 years. The lack of clinical treatments point to an urgent need for mechanistic studies of HFpEF pathogenesis. The cardiac remodeling and dysfunction in HFpEF is driven by comorbidities such as obesity, diabetes, arterial hypertension and endothelial dysfunction which lead to systemic inflammation. Here we report that, in mice, simultaneous metabolic and hypertensive stress, through the combination of high fat diet (HFD) and constitutive nitric oxide (NO) synthase inhibition by administration of N[w]-nitro-l-arginine methyl ester (L-NAME) recapitulates systemic and cardiovascular alterations of human HFpEF. The heart failure phenotype was only observed under combined treatment with HFD and L-NAME and was associated with molecular changes within the cardiomyocytes. Specifically, the spliced form of X-box binding protein 1 (Xbp1s), a downstream effector of the unfolded protein response (UPR), exhibited a significant reduction in the HFpEF model hearts and cardiomyocytes. This decrease in Xbp1s was also observed in human HFpEF hearts. Reduction in Xbp1s was caused by increased inducible NO synthase (iNOS) activity and S-nitrosylation of IRE1a, which ultimately led to a defect in XBP1 splicing activity. Importantly, cardiomyocyte-specific inducible overexpression of Xbp1s partially ameliorated diastolic dysfunction, exercise intolerance and pulmonary congestion in HFpEF mice. We have developed a novel preclinical model of HFpEF, unveiling iNOS-driven dysregulation of Xbp1s as a crucial mechanism of cardiomyocyte dysfunction. These results demonstrate the essential role of protein homeostasis dysregulation in HFpEF cardiomyocytes and suggest that inhibiting iNOS activity may be a viable therapeutic strategy in this condition
