Abstract The overarching goal of this proposal is to uncover novel HUMAN-SPECIFIC molecular mechanisms by which human long non-coding RNA (lncRNA) HL6 facilitates lipid droplet (LD) transport in human cardiomyocytes (CMs) and manifests a cardioprotective role against metabolic syndrome associated cardiomyopathy. ~70% energy of adult human heart is obtained from oxidation of long chain fatty acids (FAs). Balanced lipid metabolism is crucial for heart function. Compromised lipid metabolism have been widely observed in cardiomyopathies associated with metabolic syndromes, such as obesity and diabetes. The distinct features of obese and diabetes associated cardiomyopathy are increased incidence of heart failure together with extensive intramyocyte lipid droplet accumulation. LD consists of a neutral lipid core surrounded by a phospholipid monolayer, which plays a critical role in excess fatty acid storage and mobilization. However, overwhelmed intramyocyte LDs can cause cardiac lipotoxicity and dysfunction in both human patients and rodent models of obesity and diabetes. Although ample evidence suggests that LD accumulation in CMs could be due to impaired LD transport, the cellular system for LD transport in mammalian heart remains elusive. Particularly, the human-specific LD transport system of CMs and its implication in metabolic syndrome associated cardiomyopathy and heart failure remain unknown. Recently, we identified a human lncRNA, Heart LncRNA 6 (HL6), which is highly and specifically expressed in human CMs and downregulated in human type 2 diabetic myocardium. HL6 was knocked out (HL6KO) in human iPSCs (hiPSCs) by using CRISPR/Cas-9. We found HL6 deficiency led to extensive LD accumulation, defective LD transport to mitochondria, swollen mitochondria with impaired function, and enhanced CM death in HL6KO hiPSC-CMs verse WT hiPSC-CMs. Intriguingly, HL6 binds LDs and cytoskeleton in hiPSC-CMs. Additionally, transgenic HL6 overexpression significantly mitigated high fat diet (HFD)-induced lipid accumulation and cardiac dysfunction of mouse heart. Therefore, we hypothesize that HL6 plays an indispensable role in LD transport to mitochondria in human CMs, and gain-of-HL6 manifests a cardioprotective role against metabolic syndrome associated cardiomyopathy. Two specific aims are proposed: Specific Aim 1: Decipher the indispensable role of HL6 in lipid droplet transport of human CMs. Specific Aim 2: Determine the cardioprotective role of HL6 in metabolic syndrome associated cardiomyopathy.