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When depolarized from typical resting membrane potentials (V(rest) approximately -90 mV), cardiac sodium (Na) currents are more sensitive to local anesthetics than brain or skeletal muscle Na currents. When expressed in Xenopus oocytes, lidocaine block of hH1 (human cardiac) Na current greatly exceeded that of mu1 (rat skeletal muscle) at membrane potentials near V(rest), whereas hyperpolarization to -140 mV equalized block of the two isoforms. Because the isoform-specific tonic block roughly parallels the drug-free voltage dependence of channel availability, isoform differences in the voltage dependence of fast inactivation could underlie the differences in block. However, after a brief (50 ms) depolarizing pulse, recovery from lidocaine block is similar for the two isoforms despite marked kinetic differences in drug-free recovery, suggesting that differences in fast inactivation cannot entirely explain the isoform difference in lidocaine action. Given the strong coupling between fast inactivation and other gating processes linked to depolarization (activation, slow inactivation), we considered the possibility that isoform differences in lidocaine block are explained by differences in these other gating processes. In whole-cell recordings from HEK-293 cells, the voltage dependence of hH1 current activation was approximately 20 mV more negative than that of mu1. Because activation and closed-state inactivation are positively coupled, these differences in activation were sufficient to shift hH1 availability to more negative membrane potentials. A mutant channel with enhanced closed-state inactivation gating (mu1-R1441C) exhibited increased lidocaine sensitivity, emphasizing the importance of closed-state inactivation in lidocaine action. Moreover, when the depolarization was prolonged to 1 s, recovery from a "slow" inactivated state with intermediate kinetics (I(M)) was fourfold longer in hH1 than in mu1, and recovery from lidocaine block in hH1 was similarly delayed relative to mu1. We propose that gating processes coupled to fast inactivation (activation and slow inactivation) are the key determinants of isoform-specific local anesthetic action.

The cardiomyocyte cytoskeleton, composed of rigid and elastic elements, maintains the shape of an elongated cylinder with an eccentrically-ellipsoidal cross-section, even during contraction-relaxation cycles. Mitochondria are micron-sized fluid-filled passive spheres distributed throughout the cardiomyocyte in a crystal-like lattice in pairs sandwiched between the sarcomere contractile machinery, longitudinally and radially; thus, their shape represents the balance of forces in 3D extant at any given moment. We developed a novel method to examine the average deformation of mitochondrial dimensions in 3D, in response to cardiomyocyte contraction and relaxation, to understand how dynamic forces are balanced inside the cardiomyocytes. The optical contrast provided by the periodic lattice of myofilaments alternating with rows of mitochondria was analyzed by examining the appropriate peaks in the frequency spectrum image along the respective cardiomyocyte axes. This technique enables precise resolution of changes in dimension of ∼1% in ∼1 µm (long axis) structures with a time resolution of 8 msec.During active contraction (1 Hz stimulation) the mitochondria deform along the length-and width-axes with similar time-to-peak deformation and 50% and 90% deformation duration characteristics in both sarcomere and mitochondrial structures. However, significant deformation anisotropy was observed between the orthogonal short (i.e., width & depth) axes of mitochondria during electrical stimulation. Interestingly, the same degree of deformation anisotropy was found between the myocyte orthogonal short axes during the same electrical stimulation; therefore, the mitochondria reflect the overall cell behavior, and the apparent stiffness and stress/strain characteristics of the cytoskeleton differ appreciably between the cardiomyocyte orthogonal short axes. This method may be applied to obtaining a better understanding of the dynamic force-balance inside cardiomyocytes and of changes in the cytoskeleton spatial stiffness characteristics that may accompany aging or pathological conditions.