All animal studies were conducted in accordance with institutional guidelines and the protocol was reviewed and approved by the Animal Care and Use Committee at the University of Miami Miller School of Medicine (UMMSM). UMMSM has an Animal Welfare Assurance (A-3224-01, effective July 11, 2007) on file with the Office of Laboratory Animal Welfare (OLAW), National Institutes of Health. The generation and characterization of transgenic (Tg) mice used in this study and Tg protein expression profiles have been described earlier (Kazmierczak et al., 2009, 2013; Muthu et al., 2011). Previously produced Tg-WT lines, L1, L3, and L4 expressing 88, 30, and 77% of WT-ELC (UniProtKB: P08590) and Tg-A57G lines, L1, L2, and L5 expressing 80, 55, and 75% of A57G-mutant, respectively were used in this study. As for Tg-Δ43, two lines were used for this study, one previously generated expressing 40% of Δ43 protein (L9), and the second line expressing 55% transgene which was generated by cross breeding of existing mice (L8 × L8) in order to increase transgenic protein expression (Kazmierczak et al., 2009). The percent of protein expression indicates the amount of replacement of the endogenous mouse cardiac ELC by human ventricular WT (UniProtKB: P08590) or its two mutants. In all experiments Tg-A57G and Tg-Δ43 mice were gender and age matched with Tg-WT controls (Kazmierczak et al., 2009; Muthu et al., 2011; Kazmierczak et al., 2013).

Chronic 4 week-long training by swimming of Tg-WT, Tg-A57G, and Tg-Δ43 mice was performed in water at 30–32°C (to avoid thermal stress). Initial swim time was set as 10 min, thereafter gradually increasing until 90 min sessions were reached. The 90-min training schedule was continued twice a day (separated by 4–5 h), 7 days a week, for 4 weeks. This protocol was demonstrated to be highly effective in promoting physiological hypertrophy in mice (Evangelista et al., 2003; Galindo et al., 2009).

After euthanasia, the hearts from 3 to 5-month-old Tg-Δ43, Tg-A57G, and Tg-WT mice were excised, weighed and immersed in 10% buffered formalin. Slides of whole mouse hearts were prepared at the Histology Laboratory (University of Miami Miller School of Medicine, Miami FL). The paraffin-embedded longitudinal sections of left ventricles (LV) of H&E (hematoxylin and eosin) and Masson's trichrome stained hearts were examined for overall morphology and fibrosis using a Dialux 20 microscope, 40×/0.65 NA (numerical aperture) Leitz Wetzlar objective and an AxioCam HRc (Zeiss) as described previously (Kazmierczak et al., 2009, 2013; Muthu et al., 2011).

The papillary muscles of the left ventricles from 3 to 5 months old Tg-A57G L1 mice, Tg-Δ43 (L9 and inbred cross of L8) and Tg-WT (L1, L3, and L4) mice were isolated, dissected into small muscle bundles and chemically skinned in 50% glycerol and 50% pCa 8 solution (10⁻⁸ M [Ca²⁺], 1 mM free [Mg²⁺] (total MgPr (propionate) = 3.88 mM), 7 mM EGTA, 2.5 mM [Mg-ATP²⁻], 20 mM Mops, pH 7.0, 15 mM creatine phosphate and 15 units/ml of phosphocreatine kinase, ionic strength = 150 mM adjusted with KPr) containing 1% Triton X-100 for 24 h at 4°C. Then the bundles were transferred to the same solution without Triton X-100 and stored at −20°C for 4–6 days (Kazmierczak et al., 2012).

Small muscle strips ~1.4 mm in length and 100 μm in diameter were isolated from a batch of glycerinated skinned mouse papillary muscle bundles and attached by tweezer clips to the force transducer of the Guth Muscle Research System (Heidelberg, Germany). They were placed in a 1 ml cuvette and skinned in 1% Triton X-100 dissolved in pCa 8 buffer for 30 min, as described in Kazmierczak et al. (2012). Then they were rinsed in pCa 8 and their length adjusted to remove the slack. This procedure results in sarcomere length (SL) ~2.1 μm as judged by the first order optical diffraction pattern as described in Muthu et al. (2011); Wang et al. (2013a,b); Huang et al. (2014). Muscle strips were then tested for steady state force development in pCa 4 solution (composition is the same as pCa 8 buffer except the [Ca²⁺] = 10⁻⁴ M). All experiments were carried out at 21°C. Maximal steady state tension measured in pCa 4 solution was expressed in Newtons per cross section of the muscle strip (kN/m²). The measurement of diameter was taken at ~3 points along the muscle strip length with an SZ6045 Olympus microscope (zoom ratio of 6.3:1, up to 189x maximum magnification) and averaged (Muthu et al., 2012).

After the initial steady state force was determined, muscle strips were relaxed in pCa 8 buffer and then exposed to solutions of increasing Ca²⁺ concentrations from pCa 8 to 4 (Dweck et al., 2005). Steady-state force was measured in each “pCa” solution followed by relaxation in pCa 8 solution. Data were analyzed using the Hill equation (Hill et al., 1980) where “[Ca²⁺]₅₀ or pCa₅₀” is the free Ca²⁺ concentration which produces 50% maximum force, and “n_H” is the Hill coefficient.

Mouse cardiac myofibrils were prepared according to the method described previously (Kazmierczak et al., 2013). Briefly, after euthanasia, the hearts from 3 to 5 months old transgenic mice were isolated and frozen in liquid nitrogen and stored at −80°C until processed. For the preparation of myofibrils the tissue was thawed in the CMF (cardiac myofibril) buffer consisting 5 mM NaH₂PO₄, 5 mM Na₂HPO₄(pH 7.0), 0.1 mM NaCl, 5 mM MgCl₂, 0.5 mM EGTA, 5 mM ATP, 5 nM microcystin, 0.1% Triton X-100, 20 mM NaF (phosphatase inhibitor), 5 mM DTT and 1 μl/ml protease inhibitor cocktail (Sigma-Aldrich Corp., St. Louis, MO, USA). The tissue was then homogenized in a Mixer-Mill MM301 (Retsch) until homogenous. The homogenate was centrifuged for 4 min at 8000 g and the supernatant was discarded. After centrifugation, the pellets were left on ice for 4 min. This step was repeated three times until the pellet turned white. The pellets were then resuspended in the CMF buffer and the myofibrils were subsequently dissolved in Laemmli sample buffer and loaded on 15% SDS-PAGE. The thick and thin filament proteins were detected by Coomassie brilliant blue staining. Band intensities were measured using Image J software and ratios of total myosin regulatory light chain (RLC_tot) to tropomyosin (RLC_tot./Tm) and RLC_tot./TnI (troponin I) were determined.

Mouse cardiac myofibrils were used to determine sarcomeric protein phosphorylation. After separation of the samples on 15% SDS-PAGE Pro-Q Diamond phosphoprotein gel stain reagent (Invitrogen) was used (as described in the manufacturer's manual) to assess phosphorylation of troponin (TnT, TnI) and myosin RLC. The total protein was further detected in the same gel using the Coomassie brilliant blue staining. Myofilament protein phosphorylation ratio (ProQ) was calculated relative to the corresponding Coomassie brilliant blue staining (ProQ/Coomassie) using Image J software.

Total RNA was isolated from ventricles of sedentary and exercised Tg-WT, Tg-A57G, and Tg-Δ43 mice and converted to double stranded cDNAs using Random Primers and a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) as described earlier (Kazmierczak et al., 2009). Quantitative PCR was conducted using SYBR Green I chemistry with gene-specific Quantitect Primer Sets (Qiagen) for murine: ANP (atrial natriuretic factor: NM_008725), BNP: NM_008726), Myh6 (α-myosin heavy chain, cardiac: NM_010856), Myh7 (β-MHC: NM_080728), ColVIIIa (collagen type 8a, NM_007739), and ATP2a2 (NM_001110140.3) which encodes two Ca²⁺-transporting ATPase isoforms, SERCA2a (cardiac) and SERCA2b (non-muscle). SERCA2a is the predominant Ca²⁺ pump in the myocardium (Vangheluwe et al., 2003). All reactions were performed in triplicate and run using BIO-RAD iQ5 Multicolor Real-Time PCR Detection System with the following cycle parameters: cycle of 50°C (2 min) followed by 95°C (10 min), 40 cycles of 95°C (15 s) followed by 60°C (1 min). Raw data were analyzed using the BIO-RAD CFX Manager Software, and fold change in expression of each gene was calculated using the relative quantification (RQ) ΔΔCt method with the levels of GAPDH (glyceraldehyde-3-phosphate dehydrogenase: NM_008084) as the normalizer gene (Kazmierczak et al., 2009).

All data were expressed as mean ± s.e.m. Statistical comparisons were performed using ANOVA or independent t-test (Sigma Plot 11; Systat Software, San Jose, CA). P-values <0.05 indicated statistically significant differences.

The evaluation performed for sedentary animals shows normal gross morphology for all tested 3–5 month-old male mice (Tg-WT, Tg-Δ43, and Tg-A57G) (Figure 1A). In our earlier study on sedentary Tg-Δ43 mice, we observed cardiac hypertrophy in mice ~7 months of age while the hearts of ~2 month-old Tg-Δ43 animals were indistinguishable from control Tg-WT mice (Kazmierczak et al., 2009). As we confirmed later, the hypertrophy in Tg-Δ43 mice appeared to be age dependent and profound hypertrophy was observed in ~12 month-old Tg-Δ43 mice compared with age matched Tg-WT hearts (Muthu et al., 2011). Thus, the lack of abnormal heart growth observed in 3–5 month-old sedentary Tg-Δ43 mice (Figure 1A), is not surprising and suggests that Tg-Δ43 mice have to be at least 7 months of age to develop cardiac hypertrophy. Regarding sedentary Tg-A57G mice, similar to what we reported earlier (Muthu et al., 2011), no cardiac growth could be observed in 3–5 month-old mice (Figure 1A) or the mice as old as ~12 months of age (Muthu et al., 2011). However, exercised 3–5 month-old Tg-A57G animals do show cardiac hypertrophy while the hearts of Tg-Δ43 mice were comparable in size to Tg-WT controls (Figure 1B).

Histology examination of animals subjected to strenuous exercise is presented in Figure 2. H&E and Masson's trichrome stained left ventricular (LV) sections from Tg-Δ43 hearts revealed that the hearts of exercised 3–5 month-old mice demonstrated no tissue abnormalities, myofilament disarray or fibrosis compared to Tg-WT controls. In contrast, the evaluation of LV heart sections from Tg-A57G mice showed occurrences of fibrosis, especially in the interventricular septum compartment (Figure 2). This is in accord to previously reported pathological morphology in sedentary Tg-A57G mice, which included extensive disorganization of myocytes and interstitial fibrosis (Muthu et al., 2011; Kazmierczak et al., 2013).

Hypertrophic remodeling in Tg-Δ43 and Tg-A57G animal models was studied by looking at mutation-induced alterations in cardiac contractile proteins (α- to β-MHC switch), expression of the fetal genes such as ANP and BNP. We also examined whether cardiac remodeling in these mice involved changes in the calcium handling proteins such as SERCA2a. Studies of gene expression using real-time PCR in mouse myocardium were performed for sedentary and exercised Tg animals and the results are presented in Figures 3, 4, respectively. For sedentary Tg-A57G mice, the expression of sarcoplasmic ATP2a2 was higher compared with sedentary Tg-WT (~1.6 fold). The upregulation of mRNA SERCA2a in Tg-A57G mice (Figure 3) may translate to a faster relaxation of the A57G myosin cross-bridges and potentially lower contractile force generation in Tg-A57G mice, what is actually observed (Figure 5A). For sedentary Tg-Δ43 mice the expression levels of ANP and BNP were higher compared with sedentary Tg-WT (2- and 2.4 fold, respectively) (Figure 3).

This upregulation of both natriuretic peptides in Tg-Δ43 mice most likely manifests their protective role against excessive cardiac remodeling and preventing uncontrolled myocardial growth of Tg-Δ43 hearts (Tsuruda et al., 2002; Gardner, 2003).

To evaluate a combined effect of strenuous exercise and ELC mutation on heart remodeling we have also studied the molecular mechanisms and signaling pathways that change due to exercise. Figure 4 presents the gene expression analysis using real-time PCR in mouse myocardium performed for exercised Tg ELC animals. In Tg-A57G mice the exercise training reactivated the fetal gene program and upregulation in the β-MHC, ANP and BNP was observed. Expression of ANP and BNP was respectively 1.9- and 2.1 fold higher compared with exercised Tg-WT (both differences statistically significant P < 0.05). Additionally, upregulation of collagen VIIIa (1.9 fold change, significant P < 0.05) and β-MHC (not significant) and downregulation of α-MHC was observed for exercised Tg-A57G compared with exercised Tg-WT mice (Figure 4). On the other hand, exercised Tg-Δ43 mice showed a 0.6-fold lower expression level of ANP (P < 0.05) and no change in BNP marker compared to exercised Tg-WT controls (Figure 4). Changes in gene expression observed in Tg-A57G mice in response to strenuous exercise represent a pathologic type of remodeling occurring in the disease mouse model. In contrast, lack of changes in expression levels of BNP, β-MHC and collagen VIIIa in exercised Tg-Δ43 mice represents an adaptive response to exercise and non-pathological remodeling in Tg-Δ43 animals.

The data from functional measurements assessed in skinned mouse papillary muscle strips from Tg-Δ43 and Tg-A57G animals are presented in Figure 5, and the values of maximal force and pCa₅₀ are summarrized in Table 1. Different pattern of contractile force generation and the Ca²⁺ sensitivity of force was observed betweenTg-A57G and Tg-Δ43 mice. Similar to our earlier study (Kazmierczak et al., 2013), sedentary Tg-A57G mice showed the lowest maximal force (~20% lower vs. sedentary Tg-WT) and a slightly increased Ca²⁺ sensitivity of force (ΔpCa₅₀ ≅0.04 vs. sedentary Tg-WT), both changes statistically significant. As we demonstrated earlier and in this study, sedentary Tg-A57G animals showed reduced maximal (pCa 4) force and increased Ca²⁺ sensitivity following the pattern of pathological hypertrophy (Muthu et al., 2011; Kazmierczak et al., 2013) (Figures 5, 6). Contrary, maximal force for sedentary Tg-Δ43 mice was similar to that of Tg-WT and the Ca²⁺ sensitivity was not changed (ΔpCa₅₀ ≅0.02; not statistically significant) compared to Tg-WT controls. The previously reported reduction in steady-state force in ~2 month-old and ~7 month-old Tg-Δ43 mice (Kazmierczak et al., 2009) was likely associated with a decrease in the myosin content observed in these animals vs. the lack of changes in myosin expression seen in the current investigation (Figure 7).

In response to exercise, all transgenic animals showed a higher level of maximal force per cross-section of muscle strip vs. their sedentary controls (Table 1). Similarly to the direction of changes observed for sedentary animals, in response to swimming, the maximal force for Tg-Δ43 was similar while that for Tg-A57G was significantly reduced (~20% lower, P < 0.005) compared to exercised Tg-WT. Additionally, for exercised Tg-A57G, the Ca²⁺ sensitivity of force was significantly lower compared to sedentary Tg-A57G mice (ΔpCa₅₀ ≅0.08) (Figures 5B, 6 and Table 1). Tendency toward desensitization of myofilaments to Ca²⁺ observed in Tg-A57G mice (Table 1) may represent a key step in the signaling pathway for the pathological hypertrophic remodeling in this animal model of HCM. As expected, there were no significant changes in the Ca²⁺ sensitivity of force between exercised and sedentary Tg-WT mice. Likewise, no significant changes in the Ca²⁺ sensitivity were present between the exercised and sedentary Tg-Δ43 mice (Table 1). No changes in the cooperativity (Hill coefficient) values for all groups of mice (n_H = 2.6 ± 0.3) were noted (Figure 6).

As mentioned above, the reduced level of maximal contractile force measured in our previous study for sedentary Tg-Δ43 mice was most likely associated with deficiency in myosin content detected in the myocardium of Tg-Δ43 vs. Tg-WT mice (Kazmierczak et al., 2009). Therefore, myosin content in sedentary and exercised animals was assessed in this study and correlated with the ability of the myocardium to develop contractile force. Figure 7 presents the ratios of RLC_tot./Tm and RLC_tot./TnI determined in cardiac myofibrillar preparations from sedentary and exercised Tg-WT, Tg-A57G, and Tg-Δ43 mice. Compared to respective Tg-WT myofibrils (100%), the ratios of RLC_tot./Tm and RLC_tot./TnI obtained for sedentary and exercised Tg-A57G mice were similar to Tg-WT indicating no change in myosin expression in all tested animals (Figure 7). Therefore, the lower maximal force observed for the A57G mutation in this study is not due to the lower myosin content but due to defective function of the A57G-myocardium. The values of RLC_tot./Tm and RLC_tot./TnI ratios obtained for sedentary and exercised Tg-Δ43 mice were also very similar to Tg-WT (~110%), indicating no previously observed deficiency of myosin in the hearts of Tg-Δ43 animals (Kazmierczak et al., 2009).

The force-generating capacity of sarcomeres has been known to be tightly regulated by sarcomeric proteins phosphorylation (Sweeney et al., 1993; Sadayappan et al., 2009; Nixon et al., 2012). To test whether the ELC mutation (Δ43 or A57G) and/or exercise had any effect on phosphorylation of sarcomeric proteins, we have examined phosphorylation of myosin binding protein C (MyBP-C), Tm, troponin (TnT and TnI) and myosin RLC in mouse cardiac myofibrils using the Pro-Q Diamond staining. As shown in Figure 8, no significant changes in endogenous MyBP-C, Tm, TnT, TnI, or RLC phosphorylation were found in sedentary or exercised Tg-Δ43 or Tg-A57G animals compared to Tg-WT mice. The lack of the effect of exercise on protein phosphorylation is somewhat surprising but has been observed by other laboratories. No effect of exercise on phosphorylation of myosin RLC in the ventricles or atria of mice was found by Fewell et al. (1997). Likewise, no changes in phosphorylation of titin and TnI in mice upon exercise training were recently reported by the Granzier group (Hidalgo et al., 2014). These results suggest no immediate molecular contacts between the ELC protein and the phosphorylation domains of MyBP-C, Tm, TnT, TnI, or RLC.

Since the ELC is a highly essential element of the myosin molecule, its structure and the interaction with the MHC and actin play important roles in the sarcomeric assembly and force production (Hernandez et al., 2007). This investigation was designed to look at two different cardiac phenotypes induced by structural alterations in the myosin ELC in mouse models of pathologic (Tg-A57G) and physiologic (Tg-Δ43) HCM. The effects of both ELC mutations on heart function and cardiac remodeling are summarized in Figure 9. Data obtained for sedentary and exercised Tg-A57G and Tg-Δ43 animals show clear differences between these two phenotypes. Consistent with our earlier reports, we monitored significantly compromised contractile function (lower maximal force and higher Ca²⁺ sensitivity) in sedentary Tg-A57G mice, which coincided with upregulation of the calcium handling protein SERCA2a compared to sedentary Tg-WT animals. It has been reported that in animal models, severe compensated hypertrophy in response to pressure overload is accompanied by a large decrease in the mRNA and protein content of SERCA2a (Buttrick et al., 1994; Rockman et al., 1994). However, in rats with moderate cardiac hypertrophy, the level of SERCA2a mRNA or protein was unaltered or even upregulated (Rockman et al., 1994; Arai et al., 1996). SERCA2a is a key protein responsible for maintaining a balanced concentration of Ca²⁺ during the cardiac cycle and it controls the transport of Ca²⁺ to the SR during relaxation. The upregulation of mRNA SERCA2a in Tg-A57G mice (Figure 3) may translate to an increased protein activity, which would lead to faster relaxation of the A57G myosin cross-bridges and potentially lower contractile force generation in Tg-A57G myocardium (Figure 5A).

These adverse effects were not present in sedentary Tg-Δ43 mice, the model of non-pathological cardiac phenotype. The maximal force and Ca²⁺ sensitivity values were similar to those determined for sedentary Tg-WT mice, indicating a normal heart function in Tg-Δ43 animals. As we reported earlier (Kazmierczak et al., 2009, 2013; Muthu et al., 2011) both mouse models demonstrated cardiac hypertrophic growth, but the pathological features were only observed in sedentary A57G animals (Kazmierczak et al., 2009; Muthu et al., 2011; Kazmierczak et al., 2013). Discrepancies exist regarding the lack of reduction in maximal force observed in Tg-Δ43 mice in the current study (Table 1) and the previously reported decrease in steady-state force in Tg-Δ43 mice (Kazmierczak et al., 2009). The reason for a lower force reported in Kazmierczak et al. (2009) was an approximately 30% deficit in myosin cross-bridge content in the myocardium of Tg-Δ43 mice (Kazmierczak et al., 2009). This deficit in myosin content was not observed in the current work (Figure 7), indicating a compensatory response in transgenic Tg-Δ43 mice over time. Consequently, the lack of abnormalities in myosin expression was most likely responsible for no changes in force per cross-section of muscle seen in Tg-Δ43 mice in the current investigation (Table 1). Additionaly, recently published data utilizing the same Tg-Δ43 mouse model, showed only a slight (and not significant) decrease in maximal tension between Tg-WT and Tg-Δ43 (Michael et al., 2013; Wang et al., 2013a).

Sedentary Tg-Δ43 mice also showed an upregulation of ANP and BNP (Figure 3). Upregulation of these transcripts is considered a highly conserved feature of ventricular hypertrophy (Lee et al., 1988; Dagnino et al., 1992; Buttrick et al., 1994; Nakagawa et al., 1995), but there are contradictory reports in the literature on the correlation of upregulation of natriuretic peptides with physiological hypertrophy that can be induced by various training programs in animals. Some publications report an upregulation (Buttrick et al., 1994; Allen et al., 2001), while some report no change (Azizi et al., 1995; Jin et al., 2000) or downregulation (Diffee et al., 2003) of ANP and/or BNP expression in these animal models. Both natriuretic peptides were shown to function as modulators of cardiac hypertrophy preventing uncontrolled myocardial growth and regulating ventricular remodeling secondary to compensatory hypertrophy (Tsuruda et al., 2002; Gardner, 2003). It is therefore possible that in the case of sedentary Tg-Δ43 mice, mild upregulation of ANP and BNP manifests their protective role against excessive cardiac remodeling and controlling the extent of cardiac hypertrophy.

There are some discrepancies between our earlier study and current investigation on the hypertrophic growth in sedentary Tg-Δ43 mice. In Kazmierczak et al. (2009), we presented the gross morphology images of young (~2 mo-old) and adult (~7 mo-old) Tg-Δ43 animals. The hypertrophy was only noted for the older animals. The hearts of young Tg-Δ43 mice were indistinguishable from age matched control Tg-WT mice. In the current study, the hearts of 3–5 month-old sedentary Tg-Δ43 mice were not much different than those of age matched Tg-WT controls (Figure 1A). These data suggest that hypertrophy in Tg-Δ43 mice is an age dependent process and the fact that it was not present in <5 month-old Tg-Δ43 animals (Figure 1A), i.e., ~2 months before the apparent threshold when the animals reach ~7 months of age (Kazmierczak et al., 2009) is not surprising. In line with this, a profound hypertrophy in ~12 month-old Tg-Δ43 mice was recently reported by our group (Muthu et al., 2011).

Swimming exercise is known for its efficiency in inducing cardiac hypertrophy and significant increases in LV end-diastolic volumes in rats (Geenen et al., 1988). We hypothesized that strenuous exercise applied to Tg-Δ43 and Tg-A57G mice would lead to differential cardiac remodeling in these animal models displaying two distinct phenotypes of a “healthy” (Δ43) vs. “diseased” (A57G) heart. Our data on exercised animals confirmed our hypothesis. For Tg-A57G animals, we observed a significant upregulation of hypertrophic markers that were activated upon exercise initiating pathological hypertrophic remodeling in Tg-A57G mice (Figure 9). Upregulation of ANP and BNP transcripts observed in exercised Tg-A57G mice (Figure 4) manifests their sensitivity in monitoring ventricular hypertrophy (Lee et al., 1988; Dagnino et al., 1992; Nakagawa et al., 1995). ANP and BNP are released by the heart usually in response to heart failure (cardiac injury). The BNP levels in plasma have been shown to be useful in monitoring therapy for heart failure and typically a good response to effective treatment correlates with decreased concentrations of BNP (Lee et al., 2002; Yoshimura et al., 2002). Upregulation of ANP, BNP, and Collagen VIIIa was paralleled by a visible increase in heart size in Tg-A57G mice (Figure 1B), manifesting pathological hypertrophic remodeling in Tg-A57G mice (Figure 9).

Force generation and the Ca²⁺ sensitivity of force in Tg-A57G mice were significantly reduced after strenuous excise (Tables 1, 2) manifesting pathological hypertrophic remodeling. Although maximal force in these animals was slightly improved compared to sedentary mice it was still significantly lower than that monitored for other exercised mice. It is possible that strenuous exercise caused activation of the β-adrenergic pathway in Tg-A57G hearts that could lead to compromised heart performance. Decreased Ca²⁺ sensitivity in these animals compared to sedentary Tg-A57G mice might be indicative of progressing toward heart failure as observed in animal models of HF (Cazorla et al., 2005; Belin et al., 2007) and in humans (Van Dijk et al., 2009; Hoskins et al., 2010; Sequeira et al., 2013; Witjas-Paalberends et al., 2013). The presence of collagen deposits in the myocardium of Tg-A57G mice (Figure 2) indicates the propensity of the heart to increase its LV stiffness that ultimately may result in diastolic dysfunction in Tg-A57G mice (Wu et al., 2007).

A different response on the strenuous exercise was observed for the second Tg-Δ43 animal model. Function of the heart remained normal and both maximal force and the Ca²⁺ sensitivity were similar to exercised Tg-WT mice. Additionally, exercised Tg-Δ43 animals showed similar pattern of gene expression to that observed in exercised Tg-WT mice, indicating that hypertrophic remodeling of the heart was no longer present in this animal model. The lack of changes in expression levels of BNP, β-MHC, and collagen VIIIa in exercised Tg-Δ43 mice represents an adaptive response to exercise and non-pathological remodeling in Tg-Δ43 animals compared to detrimental heart remodeling observed in exercised Tg-A57G mice.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.