Parvalbumin Corrects Slowed Relaxation in Adult Cardiac Myocytes Expressing Hypertrophic Cardiomyopathy-Linked α-Tropomyosin Mutations by Pierre Coutu, Christina N. Bennett, Elizabeth G. Favre, Sharlene M. Day, and Joseph M. Metzger Circulation Research Volume 94(9):1235-1241 May 14, 2004 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. Parvalbumin expression optimization in cotransduced cardiac myocytes. Figure 3. Parvalbumin expression optimization in cotransduced cardiac myocytes. A, Strategy to optimize Parv expression. In synchronous gene transfer, the myocytes were transduced with both vectors immediately after myocyte isolation (group: A63V+PV). To reduce Parv expression to optimal concentrations while still maintaining the same level of α-Tm-A63V replacement, we generated a new group of myocytes (group:A63V+PVdel) in which α-Tm-A63V transduction was performed immediately after myocyte isolation, but gene transfer of Parv was delayed until 1.5 days later. B, Western blots showing the same amount of α-Tm-A63V replacement in both groups (A63V+PV and A63+PVdel), but a lower level of Parv in the A63V+PVdel group. C and D, Summary of the sarcomere shortening time from peak to 50% relengthening and amplitude, respectively. Results showed that the delayed strategy for Parv myocytes in addition to correcting diastolic dysfunction caused by the A63V mutation, brought contraction amplitude back to the level observed in control myocytes. Values are mean with SEM; n=48 to 56. Different from *Control and #A63V, respectively, P<0.05. Pierre Coutu et al. Circ Res. 2004;94:1235-1241 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Dual expression of parvalbumin and α-tropomyosin mutant A63V in rat cardiac myocytes. Figure 1. Dual expression of parvalbumin and α-tropomyosin mutant A63V in rat cardiac myocytes. A, Immunofluorescence detection of Parv (green) and α-Tm-Flag (red) in isolated myocytes simultaneously transduced with Parv and α-Tm-Flag recombinant vectors. Merged image (yellow) shows that nearly all myocytes express both genes. Calibration bar=100 μm. Insets are high magnifications revealing the diffuse nature of Parv expression (typical of myoplasmic soluble proteins) and the striated nature of the α-Tm-Flag (showing myofilament incorporation). B, Western blot analysis of Parv, α-Tm, and actin. For the α-Tm blots, native α-Tm is the bottom band, whereas the top band is transduced α-Tm, wild type (Tm) or mutant (A63V and A63V+PV). Slower migration of the transduced α-Tm is due to attached Flag. Level of replacement by the transduced α-Tm was ≈25% in all applicable groups (see text). Parv was detected only in the transduced groups (PV and A63V+PV). Superior vastus lateralis (SVL) muscle samples were used to quantify Parv expression. Actin was used to normalize for protein loading. PV indicates parvalbumin; A63V, α-Tm-A63V-Flag; Ctrl, Control or nontransduced; Tm, wild-type α-Tm-Flag. Pierre Coutu et al. Circ Res. 2004;94:1235-1241 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Sarcomere shortening data in transduced adult cardiac myocytes. Figure 2. Sarcomere shortening data in transduced adult cardiac myocytes. A, Representative normalized sarcomere shortening for 3 of the groups studied: Control, A63V, and A63V+PV. B through D, Summary of the time from peak sarcomere shortening to 50% relengthening (T50R; B), to 90% relengthening (T90R; C), and shortening amplitude (D). Myocytes transduced with A63V alone showed slower relaxation kinetics, and increased shortening amplitude. When coexpressed with Parv the relaxation kinetics became even faster than the control myocytes. However, the A63V+PV also showed a reduction in shortening amplitude. Values are mean with SEM; n=96 to 108. Different from *Control and #A63V, respectively, P<0.05. Pierre Coutu et al. Circ Res. 2004;94:1235-1241 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. Parvalbumin and α-Tm-E180G transgenic mice. Figure 4. Parvalbumin and α-Tm-E180G transgenic mice. A, Breeding strategy between heterozygous Parv and α-Tm-E180G transgenic mice. Offspring were nontransgenic (PV−/E180G−), expressing only one of the genes (PV−/E180G+ or PV+/E180G−), or expressing both genes (PV+/E180G+). Expected sarcomere shortening behavior is shown in the right column. Frequency of occurrence (Occ.) of each genotype was assessed for n=60 mice using PCR, and approximated the expected Mendelian inheritance. B, Protein expression was confirmed via Western blots. For α-Tm blots, the top band is the endogenous α-Tm, and the bottom band is the E180G mutant. Western blot for Parv and α-Tm were taken from different gels due to a difference in their respective antibody sensitivity (see online data supplement). Pierre Coutu et al. Circ Res. 2004;94:1235-1241 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. Myocyte sarcomere shortening and calcium fluorescence data in transgenic mice. Figure 5. Myocyte sarcomere shortening and calcium fluorescence data in transgenic mice. A, Representative normalized sarcomere shortening for myocytes from each genotype. PV−/E180G+ showed cellular diastolic dysfunction, and both groups expressing PV (PV+/E180G− and PV+/E180G+) showed very rapid relaxation kinetics. Arrows highlight the small and slower after contraction often observed in myocytes expressing both genes (PV+/E180G+). B and C, Summaries of the sarcomere shortening kinetics (B) and amplitude (C). Results show the same tendency as in the rat myocytes (see Figure 2). D, Summary of calcium fluorescence as measured with Fura-2AM. Results show little effect between the E180G expressing and nonexpressing groups, whereas a marked acceleration is observed in both groups expressing Parv. Values are mean with SEM; n=59 to 72. Different from *PV−/E180G− (control) and #PV−/E180G+ (diastolic dysfunction), respectively, P<0.05. Pierre Coutu et al. Circ Res. 2004;94:1235-1241 Copyright © American Heart Association, Inc. All rights reserved.

Figure 6. Effect of stimulation frequency on cardiac myocyte contractile performance. Figure 6. Effect of stimulation frequency on cardiac myocyte contractile performance. Summaries of sarcomere shortening amplitude (A) and time from peak (B) to 90% relengthening at 0.2 and 2.0 Hz. Values are mean with SEM; n=71 to 72 for 0.2 Hz and n=23 to 24 for 2.0 Hz. *Difference between 0.2 and 2.0 Hz data using unpaired t test, P<0.05. Pierre Coutu et al. Circ Res. 2004;94:1235-1241 Copyright © American Heart Association, Inc. All rights reserved.