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Aging is associated with diminished cardiac function in males. Cardiac excitation-contraction coupling in ventricular myocytes involves Ca influx via the Ca current (I ) and Ca release from the sarcoplasmic reticulum, which occur predominantly at t-tubules. Caveolin-3 regulates Ca t-tubular I , partly through protein kinase A (PKA), and both I and caveolin-3 decrease with age. We therefore investigated I and t-tubule Ca Ca Ca structure and function in cardiomyocytes from male wild-type (WT) and caveolin-3-overexpressing (Cav-3OE) mice at 3 and 24 months of age. In WT cardiomyocytes, t-tubular I -density was reduced by ~50% with age while surface I density was unchanged. Although regulation by Ca Ca PKA was unaffected by age, inhibition of caveolin-3-binding reduced t-tubular I at 3 months, but not at 24 months. While Cav-3OE increased Ca cardiac caveolin-3 protein expression ~2.5-fold at both ages, the age-dependent reduction in caveolin-3 (WT ~35%) was preserved in transgenic mice. Overexpression of caveolin-3 reduced t-tubular I density at 3 months but prevented further I loss with age. Measurement of Ca release Ca Ca at the t-tubules revealed that the triggering of local Ca release by t-tubular I was unaffected by age. In conclusion, the data suggest that the Ca reduction in I density with age is associated with the loss of a caveolin-3-dependent mechanism that augments t-tubular I density. Ca Ca Keywords: Caveolin-3, Excitation-contraction coupling, Ca signaling It is generally recognized that aging is associated with changes in be labile (15), and changes in both t-tubule structure and function normal cardiac function, although the cellular mechanisms underly- have been implicated in the impaired contractility observed in heart ing this remodeling remain unclear (1,2). It is becoming apparent failure (16,17). However, the effect of aging on t-tubule structure that the effects of age on the heart differ between the sexes (2). For and function is unknown. example, while the contractile amplitude of ventricular myocytes The cholesterol-binding membrane protein caveolin-3 (Cav-3) isolated from male mouse hearts were reduced by age, age did not has been suggested to contribute to t-tubule development (18,19) and affect contractility of myocytes from female mouse hearts (3,4). In also plays an important role in the localization of a striking variety of male ventricular myocytes, reduced L-type Ca current (I ) density ion channels, transporters, and signaling proteins at the sarcolemma Ca (4–7), altered ryanodine receptor (RyR) activity and slowed sarco- of cardiac myocytes (20–22), including the localization of L-type plasmic reticulum (SR) Ca uptake have been suggested to contribute Ca channels (LTCCs; and thus I ), Na-Ca exchange (NCX) and β - Ca 2 to the effects of physiological aging on excitation-contraction (E-C) adrenoceptors, to the t-tubules (23–27). It has also been suggested that coupling (8–11). Transverse (t-) tubules, invaginations of the surface Cav-3 plays a role in the constitutive regulation of I at the t-tubules Ca membrane that are central to E-C coupling (12–14), are known to (25). Recent studies have shown that Cav-3 expression declines with © The Author(s) 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/biomedgerontology/article/73/6/711/4718153 by DeepDyve user on 16 July 2022 712 Journals of Gerontology: BIOLOGICAL SCIENCES, 2018, Vol. 73, No. 6 age (28,29) and a role has been suggested for this decrease in Cav-3 expression in the development of the aged phenotype (30). We have, therefore, investigated the effect of age on t-tubule structure, I , and intracellular Ca transients, in myocytes from male Ca wild-type (WT) mice and whether cardiac-specific over-expression of Cav-3 (31) protects against the effects of aging on the heart. Methods Further details of experimental methods are provided in the Supplementary Material available online (https://academic.oup.com/ biomedgerontology). Animals All procedures were performed in accordance with UK legislation. Transgenic mice with cardiac myocyte-specific over-expression of Cav-3 (Cav-3OE) were generated using animals from Tsutsumi et al. (31) and WT C57Bl/6 littermates. Animals were kept in temperature- controlled rooms with ad libitum access to food and water. Myocyte Isolation Ventricular myocytes were isolated from the hearts of 3- and 24-month-old male WT and Cav-3OE mice. Animals were injected with heparin (500 I.U., i.p.) and 5 minutes later killed by cervical dis- location, the heart rapidly excised and myocytes isolated using our standard methods (26), and used on the day of isolation. Figure 1. Effect of age and Cav-3 over-expression on cell size and t-tubule organization. (A) Mean cell length, width and capacitance measured in intact Solutions myocytes from wild-type (WT) (black bars) and Cav-3OE (gray bars) mice The standard perfusion solution used in these experiments con- at 3 months and 24 months of age. Two-way analysis of variance (ANOVA) tained (in mM): 133 NaCl, 5 KCl, 1 MgSO , 1 CaCl , 1 Na HPO , 4 2 2 4 (age, genotype) tests yielded results as follows. Length: age p < .001, 10 D-glucose, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid genotype ns, interaction p < .01. Width: age p < .001, genotype ns, (HEPES), pH 7.4 (NaOH). During electrophysiological recordings, interaction ns. Capacitance: age p < .001, genotype ns, interaction p < .05. KCl was substituted with CsCl to inhibit K currents and the pip- (B) Corresponding data from detubulated (DT) myocytes. Length: age ns, genotype p = .03, interaction ns. Width: age p = .027, genotype ns, interaction ette solution contained (in mM): 110 CsCl, 20 TEACl, 0.5 MgCl , ns. Capacitance: age ns, genotype ns, interaction p = .049. Asterisks indicate 5 MgATP, 5 BAPTA, 10 HEPES, 0.4 GTP-Tris, pH 7.2 (CsOH). All *p < .05, **p < .01, and ***p < .001 (Bonferroni corrected post-hoc test). experiments were performed at room temperature. Where stated, 1 n/N indicated on bars. (C) Representative confocal images of t-tubules μM of TAT-tagged Cav-3 scaffolding domain (C3SD) peptide (32,33) labeled with di-8-ANEPPs. Scale bar shows 10 μ m. (D) Mean t-tubule was used as described previously (25). The C3SD peptide is thought skeleton density. (E) Mean percentage of t-tubules that were oriented along to disrupt binding of Cav-3 to its partner proteins at the scaffolding the long-axis of the cell (“longitudinal”). Asterisks and n/N as in A and B. domain (32,33). While the role of the caveolin scaffolding domain in interactions with partner proteins has been questioned (34,35), pre- 24 months of age. Age was associated with an increase in cell length treatment of cardiac myocytes with the peptide has previously been (p < .001, two-way ANOVA) and cell width (p < .001, two-way shown to inhibit Cav-3-dependent signaling compared with cells ANOVA) with the increase in length and width being greater in the treated with scrambled control peptide (25,32,33). Cav-3OE than in WT mice (increase in length: WT ~5%, Cav-3OE ~19% and width: WT ~11%, Cav-3OE ~22%). Cell capacitance, an Statistics electrical measure of cell surface membrane area, also increased with Data are expressed as mean ± SEM. Paired and unpaired t tests age in both WT and Cav-3OE cells (by ~24% and 55%, respect- or Mann–Whitney test and one- or two-way analysis of variance ively; p < .001, two-way ANOVA, Figure 1A). There was no dif- (ANOVA) were used with the Bonferroni post-hoc test where applic- ference in cell width between WT and Cav-3OE myocytes at either able. Current density-voltage relationship curves were analyzed with 3 or 24 months, whereas at 24 months, but not at 3 months, Cav- two-way repeated measures (RM) ANOVA with Bonferroni post- 3OE myocytes were longer than WT (p < .05, two-way ANOVA, hoc test. The limit of statistical confidence was p < .05. Sample sizes Bonferroni post hoc test). Mean cell width, length, and capacitance (n/N) represent the numbers of cells and animals, respectively. of detubulated cells are shown in Figure 1B. The relationship between cell membrane area and cell size is difficult to predict due to the presence of t-tubules. We there- Results fore constructed a simple geometric model cell to examine the Effect of Age and Cav-3OE on Cell Morphology expected relationship between membrane area and cell size, assum- Aging from 3 to 24 months was associated with cellular hypertrophy ing no changes in t-tubule density (for details, see Supplementary in cardiac myocytes. Figure 1A shows mean data for length and Material). In brief, myocyte geometry was approximated by a closed width of myocytes isolated from WT and Cav-3OE mice at 3 and elliptical cylinder, with t-tubules approximated by round cylinders Downloaded from https://academic.oup.com/biomedgerontology/article/73/6/711/4718153 by DeepDyve user on 16 July 2022 Journals of Gerontology: BIOLOGICAL SCIENCES, 2018, Vol. 73, No. 6 713 invaginating the cell. The model predicted a 22% increase in total membrane area of WT myocytes and a 55% increase in total mem- brane area of Cav-3OE myocytes simply as a result of the measured age-dependent hypertrophy, which agrees well with the observed increases in cell capacitance with age in the two genotypes (24% and 55%, respectively). To examine the effects of age and Cav-3OE on t-tubule struc- ture, live myocytes were stained with di-8-ANEPPS to label lipid membranes continuous with the surface sarcolemma. Representative confocal images show modest changes in t-tubule organization with age (Figure 1C). Quantification of the t-tubule skeleton showed that aging in WT and OE myocytes was associated with a 12% and 14% reduction in t-tubule density (p < .01, two-way ANOVA), re- spectively (Figure 1D), with no significant effect of Cav-3OE. This slight decrease in t-tubule density with age was not accompanied by changes in tubule orientation, as the proportion of longitudinal tubules remained the same (Figure 1E). Cav-3OE did not appear to alter tubule orientation over this age range. Taken together, these data suggest that aging is accompanied by an increase in cell width and capacitance, with a small decrease in t-tubule density. While the age-related hypertrophy was augmented slightly in Cav-3OE myocytes, Cav-3 over-expression had little effect on t-tubule morphology at either age and did not ameliorate the effect of age on cell and t-tubule morphology. Effect of Age and Cav-3OE on I Ca Since Cav-3 has been implicated in localization of I to the t-tubules Ca (25,27), we investigated I distribution and regulation with age and Ca Cav-3 over-expression. I was recorded from intact (Figure 2A, Ca Figure 2. Effect of age and Cav-3 over-expression on I density. (A) top) and DT (Figure 2A, bottom) myocytes from 3-month (left pan- Ca Representative records of I elicited by step depolarizations to −30, −20, −10, els) and 24-month (right panels) WT hearts. The corresponding I Ca Ca and 0 mV recorded from intact (top panels) or detubulated (DT, bottom panels) density-voltage relationships (Figure 2B) show that I density was Ca myocytes isolated from 3- and 24-month-old wild-type (WT) mice. Scale reduced with age. Absolute I in WT myocytes was not significantly Ca bars show 100 ms. (B) Mean I density-voltage relationships recorded from Ca different at the two ages (Supplementary Table 1), which suggests 3-month intact (closed circles) and DT (open circles) myocytes, and 24-month that the decrease in I density with age was primarily due to the intact (closed squares) and DT (open squares) myocytes from WT and Cav-3OE Ca increase in membrane area (by 24%, measured as cell capacitance) animals. Analysis with two-way repeated measures analysis of variance (RM ANOVA) yielded: intact myocytes, age p < .01, voltage p < .001, interaction p < without a commensurate increase in LTCC number. Assuming no .001; DT myocytes, age ns, voltage p < .001, interaction ns. (C) Mean I density Ca change in absolute I , either at the t-tubules or at the surface sarco- Ca at 0 mV for intact and DT (“surface”) myocytes, with estimated t-tubular I Ca lemma, the geometric model predicts that the increase in total mem- density in 3- (gray) and 24-month (black) WT myocytes. ** indicates p < .01 by brane area due to cellular hypertrophy would be associated with a Student’s t test. (D) Corresponding representative records of I intact and DT Ca greater decrease in I density at the t-tubules than at the surface myocytes isolated from 3- and 24-month-old Cav-3OE mice, to the same time Ca membrane (Supplementary Figure S1, G and H). To test this idea, scale as panel A. (E) Mean I density-voltage relationships recorded from Cav- Ca 3OE myocytes, using the same key as panel B. Analysis with two-way RM I was recorded from DT 3-month and 24-month WT myocytes. I Ca Ca ANOVA yielded: intact myocytes, age ns, voltage p < .001, interaction ns; DT density was reduced following DT at the two ages, consistent with myocytes, age ns, voltage p < .001, interaction p < .001. (F) Mean I density Ca the predominant localization of I to the t-tubules but there was Ca at 0 mV for whole cell and DT Cav-3OE myocytes, with estimated t-tubular no significant difference in I density in DT cells at the two ages Ca I density. Dashed horizontal line in (C) and (F) corresponds to t-tubular I Ca Ca (Figure 2B; Supplementary Table S2). Thus, in WT myocytes, t-tubu- density of Cav-3OE myocytes (−9.3 pA/pF). B and E: *p < .05, **p < .01, and lar I density was decreased by ~50% (p < .002, t test) while that at ***p < .001 (Bonferroni corrected post-hoc test), C: *p < .05 (Student’s t test). Ca the cell surface was unchanged with age (Figure 2C). This compares with the 23% decrease in t-tubular I density and 15% decrease in myocytes, t-tubular I density in Cav-3OE myocytes was unchanged Ca Ca surface sarcolemmal I density predicted by the model as a result of with age. This contrasts with the decrease predicted by the model on Ca cellular hypertrophy alone. Thus, the data show that age was associ- the basis of simple geometric considerations, and suggests mainten- ated with a loss of I density from the t-tubules specifically, an effect ance of I as a result of Cav-3 OE. Ca Ca that cannot be accounted for by cellular hypertrophy alone. These data also show that over-expression of Cav-3 has a dif- In intact Cav-3OE myocytes, both absolute I and cell capaci- ferent effect on I in 3-month and 24-month myocytes. I density Ca Ca Ca tance increased with age. In consequence, unlike WT myocytes, I in intact myocytes was reduced by over-expression of Cav-3 at Ca density in intact Cav-3OE myocytes was unchanged with age (two- 3 months but not at 24 months. Comparison of Figure 2C and F way RM ANOVA, age ns, interaction ns; Figure 2D, E). In DT myo- shows that the major effects of over-expression of Cav-3 were to cytes, cell capacitance and I density were not significantly different decrease t-tubular I at 3 months, and inhibit further age-associated Ca Ca with age. Figure 2F shows calculated I density at the t-tubules, decrease in t-tubular (and thus, whole cell) I density, with little Ca Ca compared to that at the cell surface, and shows that unlike in WT effect at the cell surface. Downloaded from https://academic.oup.com/biomedgerontology/article/73/6/711/4718153 by DeepDyve user on 16 July 2022 714 Journals of Gerontology: BIOLOGICAL SCIENCES, 2018, Vol. 73, No. 6 To clarify the effect of Cav-3 over-expression at 3 months, par- ticularly whether the reduction in I was Cav-3-dependent rather Ca than a result of transgenic modification, Cav-3 scaffolding domain peptide (C3SD peptide) was used (25,32). C3SD peptide interferes with the interaction of Cav-3 with its binding partners, thus reduc- ing the effect of Cav-3 over-expression. Figure 3 shows the effect of C3SD on I density measured at 0 mV in 3 months (panel A) Ca and 24 months (panel B), WT and Cav-3OE myocytes. While ap- plication of C3SD decreased I density in 3 months WT myocytes, Ca as shown previously in rat (25), it increased I density in 3-month Ca Cav-3OE myocytes. In contrast, C3SD had no effect on I density in Ca 24-month WT or OE myocytes (Figure 3B). These data show that the reduction in I in 3-month Cav-3OE myocytes was reversed with Ca C3SD, indicating that the effect of the peptide was independent of Cav-3 expression level, and that Cav-3 dependent regulation of I Ca decreased with age. Age was associated with reduced t-tubular I density in WT Ca myocytes (Figure 2C). A possible mechanism for this observation is an age-dependent reduction in constitutive PKA-induced stimulation of t-tubular LTCCs, which is also regulated by Cav-3 (25). We there- fore used the PKA-inhibitor H-89 to investigate the role of PKA in the response to age. The mean current density-voltage relationships for I recorded in the presence of 20 μmol/L H-89 from 3- and Ca 24-month WT and Cav-3OE intact myocytes are shown in Figure 3C and D arranged allow comparison of the effect of genotype in 3-month (Figure 3C) and 24-month (Figure 3D) myocytes. The same data are shown rearranged in Figure 3E and F to allow comparison of the effects of age in WT (Figure 3E) and Cav-3OE (Figure 3F) myocytes. H-89 decreased I density in all groups of cells, regardless Ca of age, presence of t-tubules or genotype (Figure 3G, H), demonstrat- ing constitutive LTCC phosphorylation in both the cell surface and t-tubular membranes, in both young and aged myocytes regardless of Cav-3 over-expression (Figure 3I, J). More importantly, in the presence of H-89, Cav-3OE persisted in decreasing I in intact 3 month, but not in 24-month myocytes, Ca while aging decreased I in WT, but not in Cav-3OE, myocytes Ca Figure 3. Effect of inhibition of Cav-3 and PKA on I density in 3- and Ca (Figure 3C–F). Figures 3I and J also show that I density was not Ca 24-month myocytes. (A) Mean I density at 0 mV in the absence (−) and Ca significantly different at the surface membrane in the four groups of presence (+) of C3SD peptide in wild-type (WT) and Cav-3OE myocytes for myocytes, suggesting that the observed changes in I occurred in myocytes from 3-month-old mice (two-way ANOVA: C3SD ns, genotype Ca the t-tubules (cf. Figure 3G, H). These changes were similar to those ns, interaction p < .001). (B) Corresponding data for 24-month-old mice (two-way ANOVA: C3SD ns, genotype ns, interaction ns). **p < .01 and observed in the absence of H-89 (Figure 2), which suggests that the ***p < .001 Bonferroni corrected post-hoc test. White text on bars in A and effects of Cav-3OE on I were not due solely to differences in PKA- Ca B represent sample sizes (n/N). (C) Mean I density-voltage relationships Ca dependent phosphorylation. recorded in the presence of H-89 from intact 3-month WT (black circles) and Cav-3OE myocytes (gray squares). Two-way repeated measures analysis Effect of Aging and Cav-3OE on Protein Expression of variance (RM ANOVA): voltage p < .001, genotype p < .05, interaction p < .001. (D) Corresponding data for 24-month myocytes. Two-way RM To investigate whether compensatory protein changes in the trans- ANOVA: voltage p < .001, genotype ns, interaction p < .01. (E and F) Data genic mice might account for these effects, we performed a prote- presented in C and D, rearranged to compare within (E) WT (two-way RM omic analysis of myocytes from WT and OE mice (Figure 4A). These ANOVA: voltage p < .001, age p < .001, interaction p < .001), or (F) Cav- data showed altered expression in only two proteins: Cav-3 and 3OE (two-way RM ANOVA: voltage p < .001, age ns, interaction ns) mice. Heat Shock Protein β1 (HSPβ1) increased by 2.9-fold (p < .01) and (G, H) Effect of PKA inhibition on mean I density at 0 mV in the absence Ca by 1.6-fold (p < .05), respectively. The mechanism underlying the (−, from Figure 2) and presence (+) of H-89 measured in intact or DT cells from 3-month and 24-month WT (G) and Cav-3OE (H) mice. Sample sizes increased expression of HSPβ1 is unclear. Expression of Cav-3 and for intact cells/hearts in control solution and in the presence of H-89 are LTCC was examined by western blotting in 3- and 24-month WT provided in Figures 2 and 3, respectively. For DT myocytes, n/N were: WT and OE myocytes (Figure 4B, C). Cav-3 expression was ~2.5-fold 3 month = 8/3; 24 months = 6/3; Cav-3OE 3 months = 7/2; 24 months = 7/2). greater in OE myocytes compared with WT cells at both ages, and *p < .05, **p < .01, and ***p < .001, Bonferroni corrected post-hoc test. aging was associated with 35% and 22% decreases in Cav-3 ex- (I) Calculated I density at 0 mV for the whole cell, surface, or t-tubular Ca pression in WT and Cav3-OE myocytes, respectively. Despite the membranes in WT myocytes in the presence of H-89. (J) Corresponding data for Cav-3OE myocytes. *p < .05, **p < .01, and ***p < .001, Student’s age-related decrease in Cav-3 expression in Cav-3OE myocytes, the t test. level of expression of Cav-3 in 24-month Cav-3OE cells was greater Downloaded from https://academic.oup.com/biomedgerontology/article/73/6/711/4718153 by DeepDyve user on 16 July 2022 Journals of Gerontology: BIOLOGICAL SCIENCES, 2018, Vol. 73, No. 6 715 Figure 5. Cav-3 and RyR protein localization in cardiac myocytes with age Figure 4. Changes in protein expression with age and Cav-3 over-expression. and Cav-3OE. (A) Representative images of Cav-3 (red) and RyR (green) (A) Proteomic analysis of cell lysates from 3 months wild-type (WT) and Cav- labeling in 3- or 24-month-old cells from wild-type (WT) or Cav-3OE mice. 3OE mice. Proteins with expression altered in excess of ±20% (marked by Scale bar indicates 10 μm. The graphs show sarcolemmal (including vertical dashed lines) are labeled: Cav-3, Heat Shock Protein β1 (HSPβ1) and t-tubules) Cav-3 (B) or RyR (C) normalized staining intensity as a function mitochondrial (m) NADP transhydrogenase. Horizontal dashed line indicates of distance from the surface sarcolemma. *p < .05 and ***p < .001 by limit of statistical confidence (p < .05). (B) Representative Western blots and two-way analysis of variance for differences between WT and Cav-3OE. mean data for Cav-3 protein expression. (C) Representative Western blots and mean data for LTCC protein expression. Mean densities are expressed normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 3-month WT and sample sizes are shown within the bars. ** indicates p < SR Ca Release, the Systolic Ca Transient and SR Ca .01, Student’s t test. Content Whole-cell Ca transients recorded from field-stimulated (0.1, 0.2, than that in 3-month WT cells (p < .05). However, Cav-3OE did not and 1.0 Hz) myocytes showed only modest changes in Ca transient alter the expression of the LTCC α -subunit at either age. Although amplitude and time course with age and Cav-3 over-expression 1c mean LTCC expression appeared to decrease by ~15% with age in (Supplementary Figures S2A and S2B). There were also no marked both groups, this was not statistically significant. differences in time-to-peak or time-to-half decay between groups: all The effect of Cav-3 over-expression on Cav-3 protein local- demonstrated the characteristic reduction in duration with increas- ization was examined by immunocytochemistry in WT and OE ing stimulation frequency, although the Cav-3OE groups show myocytes of both ages (Figure 5A). Cav-3 staining was observed slightly longer durations (Supplementary Table 3). at the surface of the cell and in regular, transverse striations with Closer examination of Ca release near t-tubules using simul- a periodicity of ~1.8 μm, near RyR staining (lower panels), which taneous measurement of membrane potential and intracellular supports the idea that the majority of Cav-3 antigenicity is at the Ca also revealed little difference between groups (Supplementary sarcolemmal membranes (including t-tubules). The intensity of Figure S2C). The upper panels of Supplementary Figure S2C the sarcolemmal Cav-3 labeling decreased from the cell surface shows the rising phase of Ca transients scanned along the line to the interior in all groups (Figure 5B). This gradient was more of a t-tubule in representative myocytes from 3- (left panels) pronounced in Cav-3OE myocytes at both ages than in the cor- and 24-month (right panels) WT and Cav-3OE myocytes. Lower responding WT myocytes, suggesting a modest (~10%) decrease panels show the time of AP upstroke (yellow), initiation of Ca in the relative amount of Cav-3 staining at the t-tubules com- release (red), and maximum rate of rise of Ca (green). Latency pared to the surface. There were no changes in RyR labeling due to the initiation (Supplementary Figure S2D) or maximum rate to age or genotype (Figure 5C). Using RyR labeling as a marker (Supplementary Figure S2E) of Ca release were not altered by of the z-disc (since its distribution was not altered between age, nor by Cav-3OE. The heterogeneity of Ca release (the dis- groups), colocalization of Cav-3 with RyR labeling tended to re- persion, or standard deviation of Ca release latencies) was also duce with Cav-3 over-expression: from 68 ± 2% (n/N = 15/3) to unaltered (Supplementary Figure S2F). The amplitude of the Ca 58 ± 3% (n/N = 19/3) in 3-month cells and 62 ± 2% (n/N = 18/3) release induced by rapid application of caffeine (10 mM), an index to 59 ± 2% (n/N = 23/3) in 24-month cells (data not shown, p < of SR Ca content, was not significantly different between 3 and .05, two-way ANOVA). These data suggest that Cav-3OE may be 24 months in WT (∆F/F = 3.6 ± 0.2, n/N = 16/3 vs 3.4 ± 0.3, associated with a mildly altered Cav-3 localization (particularly n/N = 20/3) or Cav-3OE myocytes (∆F/F = 3.2 ± 0.3, n/N = 15/3 in 3-month myocytes). vs 4.1 ± 0.4, n/N = 9/3), or between genotypes. Thus, it appears Downloaded from https://academic.oup.com/biomedgerontology/article/73/6/711/4718153 by DeepDyve user on 16 July 2022 716 Journals of Gerontology: BIOLOGICAL SCIENCES, 2018, Vol. 73, No. 6 that the observed changes in t-tubular I are accompanied by only gain of Cav-3 function can cause hypertrophy. For example, the Ca modest changes in Ca handling. hypertrophic cardiomyopathy caused by knockout of Cav-3 was associated with loss of caveolae and increased p42/p44 MAPK sign- aling (39) whereas the cardiac-specific transgenic overexpression of Discussion Cav-3, as used in the present study, results in increased numbers of caveolar signalsomes (31). The present study shows, for the first time, that the reduction in Age was also associated with an increase in the fraction of the I density of male ventricular myocytes with age occurs predom- Ca membrane in the t-tubules in both genotypes determined using cell inantly at the t-tubules. The study is also the first to investigate the capacitance (Figure 1), while imaging data revealed only a modest involvement of t-tubule structure and function, and the role of Cav- reduction in t-tubule density with age (Figure 1). The apparent dis- 3, in aging. In addition to the decreased t-tubular I density with Ca crepancy cannot be explained by differences in DT efficiency (see age, the major findings of the present study were that: (i) although Methods section). However, while at 3 months, DT cell size was not Cav-3OE augmented Cav-3 expression in both age groups, it did not markedly different from that of intact cells in either WT or Cav- prevent the reduction of Cav-3 expression with age; (ii) despite large 3OE cells, 24-month DT cells were smaller than their intact coun- decreases in Cav-3 expression with age, changes in t-tubule organ- terparts (Figure 1, Supplementary Tables S1 and S2). Cell sizes from ization and Cav-3 localization were modest; (iii) age-dependent cel- DT cells provide a measure of the surface sarcolemmal membrane lular hypertrophy was not ameliorated by transgenic overexpression capacitance. Considering Laplace’s Law, the greater wall stress of Cav-3; (iv) overexpression of Cav-3 reduced t-tubular I density Ca caused by the formamide-induced osmotic shock in hypertrophied at 3-mo, and removed the age-dependent reduction in I so that Ca cells at 24 months compared with the smaller 3-month cells may the current was maintained at 24-mo; (v) in contrast to 3 months, have resulted in increased death of the larger cells and thus selected at 24 months, I did not appear to be Cav-3 dependent, as demon- Ca smaller cells in the 24-month group. Given the larger dimensions of strated by the lack of effect of Cav-3OE and application of C3SD; WT and Cav-3OE myocytes at 24 months compared with 3 months, and (vi) neither aging nor Cav-3OE appeared to have pronounced the model predicted a 22% increase in total membrane area with effects on Ca release at steady state. age for WT myocytes and a 55% increase in total membrane area for Cav-3OE cells in the absence of any change in t-tubule density, Cav-3 Expression and Localization which agrees well with the observed increase in cell capacitance with Cav-3 protein expression was reduced with age in WT myocytes age (24% and 55%, Figure 1 and Supplementary Table S2). The cor- (Figure 4B), consistent with previous reports in mice (28,29), al- responding fraction of membrane in the t-tubules for 24-month WT though localization of Cav-3 staining at the t-tubules did not change myocytes was 45%, which also agrees well with experimental data with age. Cav-3OE did not prevent age-dependent loss of Cav-3 al- (52%, Figure 1 and Supplementary Table S2). A similar analysis for though, as might be expected, Cav-3 expression was increased at changes in Cav-3OE myocytes with age revealed a 55% increase both ages above that seen in 3-month WT (31). However, Cav-3OE in total membrane area and a fraction of membrane that is in the resulted in a steeper drop in Cav-3 staining intensity from surface t-tubules of 42%, which also agree well with those obtained experi- sarcolemma to t-tubules, suggestive of a partial disruption of Cav-3 mentally (55% and 45%, respectively, Figure 1 and Supplementary protein association with the t-tubules. Alternatively, the steeper drop Table S2). Taken together, these data suggest little change in t-tubule in staining intensity from surface to interior in Cav-3OE might be a structure with age or with over-expression of Cav-3. Although Cav-3 result of the preferential localization of overexpressed Cav-3 to the has been implicated in the development of t-tubules and the cardiac- surface membrane. The degree of colocalization of Cav-3 with RyR specific overexpression of Cav-3 has previously been reported to in- labeling appears large compared to that reported by others (36,37). crease numbers of caveolae in heart muscle (19,31,40), it is striking However, in the present study, RyR labeling was used simply as a that in the present study overexpression of Cav-3 had no effect on marker for the z-disc. Due to microscope blurring, the analysis of t-tubule morphology. Presumably other structural proteins, such as relatively un-processed confocal microscopy data in the present BIN-1, are also required for t-tubule development (41). study would have over-estimated the absolute colocalization. Thus, Over-estimation of t-tubule capacitance due to small DT the present study is not inconsistent with the previous studies of 24-month myocytes might lead to under-estimation of t-tubular Scriven et al. (2005) and Wong et al. (2013) (36,37). Nevertheless, I density. However, applying the model described above to cor- Ca our simple, but straight-forward, approach enables comparison of rect t-tubular I and capacitance results in little change in the data: Ca Cav-3 protein localization between groups (ie, age and genotype). in 3-month cells, the calculated t-tubular I density is unchanged Ca (compare Supplementary Table S1 to Figure 2C), and in 24-month Cell Morphology and t-Tubule Capacitance cells, the corrected t-tubular I density for WT and OE myocytes Ca Age was associated with cellular hypertrophy and reduction in is −8.2 ± 0.9 and −10.1 ± 1.2 pA/pF, respectively (Supplementary expression of Cav-3, consistent with previous studies (1,28,29). Table S1). Thus, the interpretation of the data is unchanged: age sig- Loss-of-function mutations and knockout of Cav-3 are also associ- nificantly decreases t-tubular I density in WT (−41%, p < .01), but Ca ated with cardiac hypertrophy, consistent with a role for loss of not in OE (ns, t test). Such consideration of the effect of cell size on Cav-3 expression in age-related hypertrophic signaling (38,39). t-tubular membrane fraction and current densities may be important However, in this study, the age-dependent hypertrophy was greater in any investigation of interventions that cause changes of cell size in myocytes from Cav-3OE than WT mice, demonstrating that and has not, to our knowledge, been considered previously. the hypertrophy was increased, and not ameliorated, by overex- pression of Cav-3. Nevertheless, the data are consistent with the Distribution and Regulation of I involvement of Cav-3 in hypertrophic signaling pathways in car- Ca The present study shows that the age-dependent decrease in I diac myocytes (20–22). Presumably, due to the intimate involve- Ca density occurs primarily at the t-tubules (Figure 2) and is associated ment of Cav-3 in hypertrophic signaling pathways, either loss or Downloaded from https://academic.oup.com/biomedgerontology/article/73/6/711/4718153 by DeepDyve user on 16 July 2022 Journals of Gerontology: BIOLOGICAL SCIENCES, 2018, Vol. 73, No. 6 717 with a decrease in Cav-3 expression (Figure 4). Cav-3 has previ- OE myocytes (Figure 3), suggesting that the reduced Cav-3 expression ously been shown to associate with LTCC and elements of the β - with age in Cav-3OE was sufficient to alleviate the small inhibitory adrenergic/cAMP-dependent pathway at the t-tubule and mediate effect of overexpression on the Cav-3/PKA-dependent pathway that PKA-dependent constitutive stimulation of t-tubular I (25,27,42). was evident at 3 months. This is consistent with the results obtained Ca Pretreatment of cells with C3SD peptide reduced I density in in the presence of C3SD, which showed no effect on I in 24-month Ca Ca 3-month WT, but not in 24-month WT cells (Figure 3), suggesting Cav-3OE cells (Figure 3). In any case, while H-89 reduced whole-cell that the reduction in I density with age is associated with the loss and t-tubule I density in both 3-month and 24-month Cav-3OE Ca Ca of a Cav-3-dependent mechanism that augments t-tubular I dens- cells, there was no difference in current densities between the two Ca ity. However, the decrease of I density was not a consequence of ages, indicating that the protective effect of Cav-3 over-expression Ca reduced constitutive PKA-dependent stimulation of I with age, against age-dependent loss of t-tubular I was independent of con- Ca Ca because application of H-89 caused a robust decrease in whole-cell stitutive regulation by PKA. and t-tubular I density in both 3-month (−62% and −58%, respect- Ca ively) and 24-month (−64% and −59%, respectively) cells. Nor was Excitation-Contraction Coupling the age-dependent decrease in I density a consequence of reduced Ca The ~50% decrease in t-tubular I density with age was not asso- Ca LTCC expression (Figure 4). Age has been associated with redistri- ciated with altered Ca release at the t-tubule in the present study: bution of Cav-3 from cholesterol-rich to cholesterol-free membranes latency between action potential upstroke and Ca release, hetero- in heart muscle, indicating a loss of caveolin from caveolae with age geneity of Ca release along the t-tubule and Ca transient properties (43). This redistribution may underlie the apparent loss of associ- were not significantly affected by age (Supplementary Figure S2 and ation of Cav-3 with LTCC and the cAMP signaling pathway in the Supplementary Table S3). This is consistent with previous studies t-tubule membrane in 24-month myocytes so that the constitutive that have shown no age-dependent differences in Ca transient ampli- regulation of I became insensitive to C3SD but retained sensitivity Ca tude or duration when cells were stimulated at frequencies similar to to PKA inhibition. However, localization of Cav-3 to the t-tubules those used in the present study, although at higher frequencies the Ca was not reduced with age (Figure 5). Moreover, the age-dependent transient may be smaller and slower with age (9,44). There were no reduction in I density was associated with an increased total mem- Ca changes in RyR distribution (Figure 5), LTCC expression (Figure 4), brane area and an increased fraction of membrane in the t-tubules or SR Ca content. Thus, the unaltered Ca release may be explained (Supplementary Table S2 and Supplementary Figure S1). by: (i) the highly nonlinear relationship between I and SR Ca re- Ca Over-expression of Cav-3 was associated with reduced t-tubular lease (45,46) so that there is effectively a functional reserve in I ; (ii) Ca I density in 3-month myocytes, suggesting that I is decreased by Ca Ca since a significant proportion of LTCCs are located outside the dyad, either inhibition or overexpression of Cav-3, and consistent with a reduction in I density in this population would have little effect Ca role for Cav-3 in determining basal t-tubule I density in myocytes Ca on CICR. The former is supported by the observation that absolute from young animals (25). The reduction of I caused by OE was not Ca t-tubular I is unaltered while fractional t-tubule area increased in Ca due to a decrease in LTCC expression (Figure 4), but might reflect myocytes from old animals (Supplementary Table S2), in contrast slightly less constitutive PKA stimulation of I since application of Ca to the decrease in absolute t-tubular I with no change in t-tubular Ca H-89 to 3-month Cav-3OE cells was associated with a smaller reduc- membrane area observed in heart failure (26). The latter is supported tion in whole-cell (−56%) and t-tubular (−47%) I compared to WT Ca by recent evidence of a role for Cav-3 in the regulation of nondyadic (see Figure 3G, H). A possible explanation is that Cav-3OE resulted LTCC in cardiac muscle (47,48). in mis-location of a fraction of the protein that, in consequence, was unable to perform its native task(s) but competed for binding part- ners with normally-located Cav-3. This idea is consistent with the Supplementary Material observation that application of C3SD peptide increases I density in Ca Supplementary data is available at The Journals of Gerontology, 3-month Cav-3OE myocytes (Figure 3). Imaging data also showed Series A: Biological Sciences and Medical Sciences online. that there may be some relocation of Cav-3 with over-expression, as Cav-3OE cells showed reduced relative Cav-3 staining intensity at the t-tubules (Figure 5). Funding Interestingly, in contrast to the decrease observed in WT myo- This work was supported by the British Heart Foundation (BHF cytes, there was no change in whole cell or t-tubular I density during Ca RG/12/10/29802 [C.H.O., A.F.J., and M.B.C.], PG/14/65/31055 [C.H.O., aging in Cav-3OE myocytes (Figure 2). The preservation of I dens- Ca A.F.J.]) and grants from the National Institutes of Health (NIH HL091071 ity at the t-tubules in 24-month Cav-3OE myocytes occurs despite a [H.H.P.], HL107200 [H.H.P. and D.M.R.], HL066941 [H.H.P. and D.M.R.], 77% increase in t-tubular surface area due to age-dependent hyper- HL115933 [H.H.P. and D.M.R.], AG052722 [H.H.P.]) and the Veterans trophy. This suggests maintenance of t-tubular I as a result of Cav-3 Ca Affairs Administration (VA Merit BX001963 [H.H.P.] and BX000783 overexpression. Despite the reduction in Cav-3 expression with age [D.M.R.]). in Cav-3OE myocytes, expression of the protein remained approxi- mately twofold greater than in 3-month WT myocytes (Figure 4). Thus, the data are consistent with the proposal that overexpression Conflict of Interest of Cav-3 protected against the age-dependent loss of LTCC func- None reported. tion from the t-tubules. 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"The Journals of Gerontology - Series A: Biological Sciences and Medical Sciences" – Oxford University Press
Published: May 9, 2018
Keywords: excitation contraction coupling; ventricular myocytes; protein overexpression
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