Background: R146G and R21C mutations in cardiac TnI are associated with hypertrophic cardiomyopathy. Results: Both mutations blunt PKA-mediated effects on weakening cTnI-cTnC interaction and accelerating myofibril relaxation. Conclusion: Both mutations result in hypercontraction and impaired relaxation, which may contribute to increased risk to traumatic heart failure. Significance: This study increases mechanistic understanding of how single amino acid mutations result in cardiac contractile dysfunction.

The plasma protein von Willebrand factor (VWF) is essential for hemostasis initiation at sites of vascular injury. The platelet-binding A1 domain of VWF is connected to the VWF N-terminally located D'D3 domain through a relatively unstructured amino acid sequence, called here the N-terminal linker. This region has previously been shown to inhibit the binding of VWF to the platelet surface receptor glycoprotein Ibα (GpIbα). However, the molecular mechanism underlying the inhibitory function of the N-terminal linker has not been elucidated. Here, we show that an aspartate at position 1261 is the most critical residue of the N-terminal linker for inhibiting binding of the VWF A1 domain to GpIbα on platelets in blood flow. Through a combination of molecular dynamics simulations, mutagenesis, and A1-GpIbα binding experiments, we identified a network of salt bridges between Asp and the rest of A1 that lock the N-terminal linker in place such that it reduces binding to GpIbα. Mutations aimed at disrupting any of these salt bridges activated binding unless the mutated residue also formed a salt bridge with GpIbα, in which case the mutations inhibited the binding. These results show that interactions between charged amino acid residues are important both to directly stabilize the A1-GpIbα complex and to indirectly destabilize the complex through the N-terminal linker.

The role of plasma phospholipid transfer protein (PLTP) in lipoprotein metabolism is poorly understood. In vitro studies suggest that PLTP influences HDL size and composition and transfers phospholipids among lipoproteins. To provide an in vivo model for studies of PLTP physiology, transgenic mice that express human PLTP were generated. Human PLTP transcripts were detected in total RNA from adipose tissue, lung, heart, and spleen of the two distinct lines (A and C) of transgenic mice. Despite minimal expression of human PLTP in the liver of these transgenic mice and similar plasma phospholipid transfer activity in transgenic and non-transgenic mice (19.1 +/- 3.1 vs 18.9 +/- 2.7 mumol/ml/h), differences in lipoprotein levels were observed between transgenic and control mice receiving the same chow diet. Male transgenic mice of line C had significantly higher HDL cholesterol than control mice (76.4 +/- 4.6 vs 71.9 +/- 7.0 mg/dl, p< 0.05) and the male transgenic mice of lines A and C had a significantly lower non-HDL cholesterol (15.1 +/- 4.1 and 15.6 +/- 4.7 vs 20.9 +/- 5.5 mg/dl, P < 0.01 and P < 0.02) and a significantly higher HDL cholesterol/non-HDL cholesterol ratio than the control mice (5.3 +/- 1.3 and 5.5 +/- 2.2 vs 3.9 +/- 1.9 mg/dl, P < 0.01 and P < 0.02). Female mice from transgenic line C had higher HDL cholesterol than control mice (64.6 +/- 4.8 vs 57.4 +/- 5.1 mg/dl, P < 0.01) while female mice from line A tended to have higher HDL cholesterol/non-HDL cholesterol ratio than control mice (5.5 +/- 3.7 vs 3.8 +/- 1.4). These observations suggest that expression of PLTP in peripheral tissues play an important role in lipoprotein metabolism. Expression of human PLTP produced a more favorable lipoprotein profile and thus, enhanced expression of PLTP could potentially retard atherosclerosis.

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