Cardiac remodeling in fish: strategies to maintain heart function during temperature Change

Abstract

Rainbow trout remain active in waters that seasonally change between 4°C and 20°C. To explore how these fish are able to maintain cardiac function over this temperature range we characterized changes in cardiac morphology, contractile function, and the expression of contractile proteins in trout following acclimation to 4°C (cold), 12°C (control), and 17°C (warm). The relative ventricular mass (RVM) of the cold acclimated male fish was significantly greater than that of males in the control group. In addition, the compact myocardium of the cold acclimated male hearts was thinner compared to controls while the amount of spongy myocardium was found to have increased. Cold acclimation also caused an increase in connective tissue content, as well as muscle bundle area in the spongy myocardium of the male fish. Conversely, warm acclimation of male fish caused an increase in the thickness of the compact myocardium and a decrease in the amount of spongy myocardium. There was also a decrease in connective tissue content in both myocardial layers. In contrast, there was no change in the RVM or connective tissue content in the hearts of female trout with warm or cold acclimation. Cold acclimation also caused a 50% increase in the maximal rate of cardiac AM Mg(2+)-ATPase but did not influence the Ca(2+) sensitivity of this enzyme. To identify a mechanism for this change we utilized two-dimensional difference gel electrophoresis to characterize changes in the cardiac contractile proteins. Cold acclimation caused subtle changes in the phosphorylation state of the slow skeletal isoform of troponin T found in the heart, as well as of myosin binding protein C. These results demonstrate that acclimation of trout to warm and cold temperatures has opposing effects on cardiac morphology and tissue composition and that this results in distinct warm and cold cardiac phenotypes.

Figure 1. The effect of acclimation temperature on ventricle size.

Cold (4°C) acclimated male rainbow trout had larger ventricle to body mass ratios compared to warm acclimated (17°C) male trout but not control (12°C) trout. Furthermore, cold acclimated male trout had larger ventricle to body mass ratios compared to female trout. Trout were acclimated for a minimum of 2 months, N = 15. Relative ventricular mass: ((heart mass / body mass) * 100). Values are mean ± SEM. Brackets, if present indicate significant differences between sexes at the same acclimation temperature. Different letters above the bars indicate significant difference between acclimation groups. Different letters within the bars for male fish indicate significant differences between acclimation temperatures (p<0.05).

Figure 2. Masson's trichrome stained sections of ventricular spongy layer from thermally acclimated rainbow trout.

(A) Cx43 staining at high magnification (63x). This image shows both the cross section of trabecular bundles (∼25 µm) and individual cardiomyocytes that make up the bundles (∼5 µm). (B) cold, 4°C, (C) control, 12°C, (D) warm, 17°C where pink/purple is muscle, blue is connective tissue and white or very pale pink is “extra bundular” space.

Figure 3. Quantification of Masson's trichrome stained sections of ventricular myocardium from thermally acclimated rainbow trout.

Representative images are provided in Figs. 2 and 3. (A) Cold acclimation caused a significant increase in bundle area in the hearts of male fish. (B) Warm acclimation caused an increase in compact layer thickness in the hearts of both male and female fish. In addition, the thickness of the compact layer in the hearts of male trout was significantly greater than that of female fish in all three experimental groups. (C) The hearts of cold acclimated male trout had significantly more connective tissue in the spongy layer than that of either control or warm acclimated male fish. (D) Cold acclimation of male trout caused an increase in connective tissue content in the compact layer compared to controls while warm acclimation of the male trout caused a decrease in connective tissue content compared to controls. For panels C and D the amount of connective tissue present in the spongy layer and compact layer is presented as arbitrary units (A.U.) representing the ratio of connective tissue present in the compartment in relation to muscle. Values are mean ± SEM. Brackets, if present indicate a significant difference between sexes within an acclimation group. Different letters above the bars indicate a significant difference between acclimation groups. Different letters within the bars, if present, indicate significant differences between acclimation temperatures when each sex is analyzed separately (p<0.05).

Figure 4. Masson's trichrome stained sections of ventricular compact layer from thermally acclimated rainbow trout.

(A) cold, 4°C, (B) control, 12°C, (C) warm, 17°C where pink/ purple is muscle, blue is connective tissue and white or very pale pink is “extra bundular” space.

Figure 5. The effects of thermal acclimation on the actomyosin Mg²⁺-ATPase activity from trout ventricle.

A) When measured at 17°C the maximal activity of cardiac AM Mg²⁺-ATPase was higher in cold acclimated trout than in warm acclimated trout. There was, however, no difference in Ca²⁺ sensitivity between acclimation groups at 17°C. (B) In contrast, when measured at 7°C there was no difference in the maximal activity of cardiac AM Mg²⁺-ATPase from all three experimental groups. Values are mean ± SEM for N = 6-9 at each temperature. A summary of this data is presented in Table 1.

Figure 6. 2D-DIGE analysis of cardiac contractile proteins from thermally acclimated trout.

Thermal acclimation did not cause a change in the isoform expression of the identified contractile proteins in the trout ventricle. There was also no detected change in phosphorylation state of any of these proteins between experimental groups. (A) Representative 2D-DIGE analysis of cardiac proteomic changes with thermal acclimation. Proteins extracted from warm and cold acclimated trout were labeled with Cy-3 (green) and Cy-5 (red), respectively. The proteins were focused in the first dimension with an 18-cm, pH range 3–10NL, IPG strip. Superimposed Cy3/ Cy5 image is shown. Protein spots identified: cMyBP-C, myomesin, cardiac troponin T (cTnT), slow skeletal TnT (ssTnt), actin, tropomyosin (Tm) and regulatory light chains (RLC) are indicated. The phosphorylation state of (B) RLC, (C) ssTnT, (D) Tm, (E) cMyBP-C or (F) myomesin were not significantly affected by thermal acclimation. The most basic spot identified in a string of proteins is labeled spot 1. (White bars – cold acclimated, grey bars –control and black bars – warm acclimated).

Figure 7. 2D-DIGE analysis of the phosphorylation state of cTnI from thermally acclimated trout.

(A) Representative 2D-DIGE analysis of cardiac troponin I (cTnI) in the cold and warm acclimated trout ventricle. Proteins extracted from cold and warm acclimated trout were labeled with Cy-5 (red) and Cy-3 (green), respectively and then focused in the first dimension with an 18-cm, pH range 7–11NL, IPG strip and 8-16% gradient SDS-PAGE for the second dimension. (B) No significant differences were observed in the phosphorylation state of cTnI (White bars – cold acclimated, grey bars –control and black bars – warm acclimated).