ABSTRACT Heart failure, which afflicts more than five million adults in the United States alone, is associated with markedly increased risk of ventricular arrhythmias and sudden death. The molecular, cellular and systemic mechanisms linking heart failure to increased sudden death risk, however, are poorly understood and, despite considerable attention and effort, risk stratifying patients remains an enormous challenge. Although numerous experimental (animal/cellular) heart failure models have been developed and extensively studied, only limited insights into human arrhythmia mechanisms have been provided. Motivated to change this and advance the field, we have undertaken a comprehensive research effort aimed at defining the mechanisms involved in the physiological regulation of membrane excitability in the human heart and the pathophysiological electrical remodeling associated with human heart failure. Utilizing the infrastructure developed in the Translational Cardiovascular Biobank and Repository at Washington University for the acquisition of non-failing and failing human hearts, we have established robust, reliable methods for the isolation and in vitro maintenance of human ventricular myocytes. Here, we utilize these unique resources to test directly the hypothesis that there are regional differences in the regulation and remodeling of three voltage-dependent conductance pathways critical for the coordinated propagation of activity through the ventricles and the maintenance of normal cardiac rhythms: the Kv4.3-encoded, fast transient, outward K+ current, Ito,f; the recently identified, novel, non-inactivating Kv current component, Iss; and, the Nav1.5-encoded voltage-gated Na+ current, INa. In aim #1, we will define the functional impact of heterogeneous Ito,f remodeling on LV action potential waveforms, and identify the molecular determinants of native Ito,f channels in non-failing human LV and of Ito,f remodeling in failing human LV. In aim #2, we will test the hypothesis that there are also transmural differences in the expression and the remodeling of Iss, and define the functional consequences of cell type-specific differences in Iss expression and remodeling on LV action potential waveforms. Experiments in aim #3 will test the hypothesis that there are transmural differences in the expression, properties and remodeling of Nav1.5-encoded INa channels, particularly the late component of INa, INa,L, in non-failing and failing human LV myocytes and define the functional impact on LV action potential waveforms of heterogeneous INa,L expression and remodeling. These studies will provide new, clinically relevant, insights into the cellular/molecular mechanisms contributing to the physiological regulation and pathophysiological remodeling of native human ventricular Ito,f, Iss and INa channels. These insights will transform the refinement of human cardiac myocyte and whole heart models and translate to novel, mechanism-based strategies to target specific cell types to reduce the risk of life-threatening ventricular arrhythmias in patients suffering human heart failure.