We performed a randomized placebo-controlled study and showed administration of atorvastatin to the β-MyHC-Q403 transgenic rabbits, early and before expression of cardiac hypertrophy, prevented development of cardiac hypertrophy over a 1-year period of observation. Prevention of cardiac hypertrophy was demonstrated at organ, cell, and molecular levels. It was associated with reduced levels of active Ras, phosphorylated p44/42 MAPK, lipid peroxides, oxidized mtDNA, and the number of TUNEL-positive cells. The findings indicate the potential utility of atorvastatin in preventing evolving cardiac hypertrophy in HCM, the most common cause of sudden cardiac death in the young and a major cause of morbidity in the elderly.13
Atorvastatin was administered early and before development of cardiac hypertrophy, as determined by echocardiographic assessment of LVMI. There were no significant differences in the baseline demographic and echocardiographic phenotypes between the nontransgenic, transgenic-placebo and transgenic-atorvastatin groups. To reduce potential biases, the data were acquired and interpreted without knowledge of the group assignment. The results are strengthened by serial and multi-level phenotypic characterization. Furthermore, the use of β-MyHC-Q403 transgenic rabbits, known to recapitulate the phenotype of human HCM, strengthens the potential application of the findings to human patients. Moreover, the dose of atorvastatin was chosen based on our previous data,6
and considering the relative potency of the 2 HMG CoA reductase inhibitors, at least, as it relates to their effects on plasma levels of low-density lipoprotein-cholesterol. We documented the effectiveness of the chosen dose in reducing plasma total cholesterol levels (≈50% reduction). Thus, we administered an effective biological dose, which is expected to inhibit generation of isoprenoid intermediates of cholesterol biosynthesis.9
Moreover, the results were concordant for strong beneficial effects on prevention of molecular, histological, and morphological phenotypes of HCM without asserting an adverse effect on cardiac function. Finally, the observed beneficial effects of atorvastatin in prevention of cardiac hypertrophy in our genetic rabbit model of human HCM are also in accord with the effects of HMG-Co A reductase inhibitors on prevention of acquired forms of cardiac and myocyte hypertrophy.7,14–16
The main antihypertrophic effects of HMG CoA reductase inhibitors have been attributed to inhibition of isoprenylation of Ras, RhoA, Rac1, and Cdc42 as well as Rac1-mediated generation of reactive oxygen species via NADH oxidase activity.7,17
We detected a significant reduction in levels of membrane-bound Ras and phosphoryated p44/42 MAPK but not in the levels of activated RhoA and Rac1. The lack of discernible differences in levels of activated RhoA and Rac1 could simply reflect the inadequate resolution of immunoblotting in detecting relatively modest changes that are expected in a genetic animal model of cardiac hypertrophy, wherein the stimulus is chronic and of relatively of low magnitude. This is in contrast to in vitro cell culture experiments, wherein the stimulus is acute, direct and potent and hence, the anticipated changes in levels of activated GTPases are greater, as shown previously.6
Thus, the data does not necessarily exclude involvement of RhoA and Rac1 GTPases or Rac1-mediated oxidative stress in mediating the antihypertrophic effects of atorvastatin. It is also noteworthy that expression levels of CAT
mRNA were reduced significantly in the hearts of β-MyHC-Q403 transgenic rabbits in the placebo group but not in atorvastatin group. Decreased expression level of CAT
mRNA has been observed in a porcine model with naturally occurring HCM.18
Catalase, which reacts very efficiently with H2
to form water and molecular oxygen, in conjunction with GPX1
are the primary responsible enzymes for the removal of H2
. Although it is plausible that reduced expression level of CAT
could provide for a mechanism for the excess levels of H2
in the hypertrophic conditions, we did not detect significant reductions in the levels of catalase protein and activity. Therefore, it is unlikely that the antihypertrophic effect of the atorvastatin was predominantly attributable to upregulation of expression of CAT
in the heart, as has been noted previously.19
Other redox-regulating proteins, such as thioredoxin (TXN
, and Cu-Zn superoxide dismutase (SOD1
) also have been implicated in regulating cardiac hypertrophy and failure.18,20,21
We detected no significant changes in the expression levels of GPX1
in the β-MyHC-Q403 rabbits. We could not determine expression levels of TXN
and SOD in the heart because of lack of reliable assays in rabbits. Finally, the number of TUNEL-positive myocytes, which reflect DNA break points, was higher in the placebo and lower in the atorvastatin, as compared with the nontransgenic group. Although TUNEL-positive cells are generally considered apoptotic cells, in the absence of a discernible evidence of DNA fragmentation or increased expression of 19 kDa caspase 3, the findings could reflect enhanced myocyte DNA synthesis in the hypertrophic state in the placebo group and reduced DNA synthesis in the atorvastatin group.
The molecular mechanisms that lead to activation of p44/42 MAPK in the β-MyHC-Q403 rabbits, a finding that is in accord with our previous data,5,6
were not determined specifically, but likely to involve multiple pathways including excess oxidative stress, which has been shown to affect post-translation oxidative modification of thiols on Ras.21
Nonetheless, the signaling pathways that could activate p44/42 MAPK are complex and interactive, encompassing Ras-dependent and –independent pathways, such as the activation of phospholipase C and protein kinase C pathways. Additional investigations would be required to delineate the molecular signaling involved in cardiac hypertrophic response in the β-MyHC-Q403 rabbits.
Administration of atorvastatin had a modest but not statistically significant effect on increased myocardial CVF and expression level of COL1A1
mRNA in the β-MyHC-Q403 rabbits. The finding appears in discord with our previous data showing normalization of myocardial CVF with simvastatin therapy in the β-MyHC-Q403 rabbits6
and those of others, showing prevention of interstitial fibrosis post myocardial infarction in rats.22
The discrepancy could reflect potential differences in the biological effects of HMG-CoA reductase inhibitors and/or the experimental design of the studies including the drug dosages. Nonetheless, the findings suggest dissociation of the antihypertrophic and antifibrotic effects of atorvastatin, either because of the differential dose effects on hypertrophy and fibrosis and/or involvement of different molecular mechanisms by which HMG-CoA reductase inhibitors exert their antihypertrophic and antifibrotic effects.
We have proposed that myocardial dysfunction is the initial functional defect caused by the β-MyHC-Q403 mutation that provokes reactive cardiac hypertrophy in HCM.3,23
In the present as well as in the previous studies, indices of global left ventricular systolic function were normal in the β-MyHC-Q403.4–6,23
The finding of normal global left ventricular systolic function does not detract from the proposed hypothesis, because myocardial contraction and relaxation abnormalities, as detected by tissue Doppler imaging, were reduced despite preserved global left ventricular systolic function.5,6,23
Impaired myocardial contraction and relaxation velocities were initially observed in the β-MyHC-Q403 transgenic rabbits and were subsequently confirmed in humans with HCM mutations but no discernible cardiac hypertrophy.24
It is also noteworthy that the left-ventricular fractional shortening was higher in 6 month-old (baseline) as compared with 18 month-old rabbits (12-month follow up) in all 3 groups. The decline was slightly greater in and statistically significant in the nontransgenic group (NTG) group. The reason(s) for the age-dependent decline in the left ventricular fractional shortening is unclear. It could be the normal physiology or could reflect an increase in body weight with aging that occurred in all three groups. However, the increased load could have a greater impact on the load-dependent indices of left ventricular function, such as the fractional shortening, in the NTG rabbits as opposed to transgenic rabbits with HCM. We note that despite the decrease, the left ventricular fractional shortening remained within the normal range in all three groups and was not considered pathological. Thus, the findings are not expected to detract from the application of the results of this study to humans with HCM.
The results of the present study showing prevention of cardiac hypertrophic response in a transgenic rabbit model of human HCM have considerable clinical implications. The clinical significance of the findings is underscored by the lack of an effective pharmacological intervention to prevent cardiac hypertrophy in human patients with HCM. Moreover, current pharmacological therapy is largely empiric and none has been shown to prevent, attenuate or reverse cardiac hypertrophy in HCM. The results, once confirmed in humans, would raise the possibility for an early intervention, using a safe and well-established class of pharmacological agents, in HCM mutation carriers to prevent the development of cardiac phenotype. Furthermore, because hypertrophy is the common response of the heart to many forms of stress and a major determinant of mortality and morbidity, regardless of the cause,25
the findings could have broader implications in treatment and prevention of many forms of cardiovascular disease.
In summary, in a randomized placebo-controlled study, we have shown that administration of atorvastatin early and before development of cardiac hypertrophy, prevented evolution of cardiac hypertrophy at organ, cell and molecular levels and reduced levels of myocardial lipid peroxides and oxidized mtDNA as well as the number of TUNEL-positive myocytes. These findings, beckon the need for clinical studies in humans carriers of HCM mutations to determine the potential beneficial effects of HMG-CoA reductase inhibitors in prevention of cardiac hypertrophy.