Absence of Type VI Collagen Paradoxically Improves Cardiac Function, Structure, and Remodeling After Myocardial InfarctionNovelty and Significance
Rationale: We previously reported that type VI collagen deposition increases in the infarcted myocardium in vivo. To date, a specific role for this nonfibrillar collagen has not been explored in the setting of myocardial infarction (MI).
Objective: To determine whether deletion of type VI collagen in an in vivo model of post-MI wound healing would alter cardiac function and remodeling in the days to weeks after injury.
Methods and Results: Wild-type and Col6a1−/− mice were subjected to MI, followed by serial echocardiographic and histological assessments. At 8 weeks after MI, infarct size was significantly reduced, ejection fraction was significantly preserved (43.9%±3.3% versus 29.1%±4.3% for wild-type), and left ventricular chamber dilation was attenuated in the Col6a1−/− MI group (25.8%±7.9% increase versus 62.6%±16.5% for wild-type). The improvement in cardiac remodeling was evident as early as 10 days after MI in the Col6a1−/− mice. Myocyte apoptosis within the infarcted zones was initially greater in the Col6a1−/− group 3 days after MI, but by day 14 this was significantly reduced. Collagen deposition also was reduced in the infarcted and remote areas of the Col6a1−/− hearts. The reductions in chronic myocyte apoptosis and fibrosis are critical events leading to improved long-term remodeling and functional outcomes.
Conclusions: These unexpected results demonstrate for the first time that deletion of type VI collagen in this knockout model plays a critical protective role after MI by limiting infarct size, chronic apoptosis, aberrant remodeling, and fibrosis, leading to preservation of cardiac function.
- cell matrix
- myocardial infarction remodeling
- nonfibrillar collagen
The extracellular matrix plays a key role in cardiac remodeling and wound repair after a myocardial infarction (MI). Patients who survive a MI normally have development of cardiac fibrosis, which contributes to the decline in cardiac function and eventual failure. Matrix turnover is critical in the days and weeks after MI; however, the functions of specific extracellular matrix components in this process remain poorly defined.1–4
In This Issue, see p 795
It has been accepted that type I and type III collagen are major constituents of the cardiac extracellular matrix that provide structural and mechanical support to the heart and act as signaling conduits between myocardial cells.2–4 Whereas the extracellular matrix field has focused on these fibrillar collagens, our recent studies have demonstrated that type VI collagen induces myofibroblast differentiation in vitro and that its deposition is enhanced in vivo after MI.5,6
Type VI collagen mutations can cause Bethlem myopathy, an age-related disease characterized by skeletal muscle weakness and limited life span.7 Type VI collagen is a nonfibrillar collagen that assembles end-to-end in a beaded filament arrangement.8 Typically interspersed with types I and III collagen, collagen VI forms a microfilament network that organizes the fibrillar collagens and anchors these to the basement membrane.9 A model for Bethlem myopathy was generated by targeted deletion of the Col6a1 gene, which causes early-onset myopathy.10,11 Although skeletal muscle defects caused by collagen type VI mutations have been described, none has determined the consequences of its absence using MI injury models. The Col6a1 null mutant mouse provides an in vivo model to determine the impact of collagen VI deficiency in skeletal muscle,12,13 as well as in other tissues such as adipose, cartilage, brain, and tendon.14–16 Here, we demonstrate that the absence of type VI collagen provides a profound beneficial effect on cardiac function and remodeling after MI.
Seventy-five male Col6a1−/− and wild-type (WT) mice in the CD-1 background at 12 to 16 weeks of age were used in this study. Detailed Methods are provided in the Online Supplement.
Surgical Induction of MI
Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (70 mg/kg), and the heart was accessed via left thoracotomy and MI was induced by permanent occlusion of the left anterior descending artery.
Histological Assessment of Apoptosis, Fibrosis, and Infarct Size
Animals were euthanized and cryosections were prepared for apoptosis assessment using TUNEL staining method as previously described.6,7 Additional hearts were embedded in paraffin and entire hearts were sectioned serially for assessment of collagen deposition using Masson trichrome, picrosirius red, and picrosirius red under polarized light. Infarct sizes were quantitated by 2,3,5-triphenyltetrazolium chloride staining.
Two-dimensional echocardiography was performed and calculations were performed offline by double-blinded reviewers using the Vevo 770/3.0 system and software (VisualSonics).
Data analysis was performed using Graphpad Prism 4.0 software (Graphpad Software). Significance was determined by ANOVA with Bonferroni post-test (P<0.05 considered significant).
Physiological Measurements of Col6a1−/− Versus WT Mice Before and After MI
The physiological features of WT and Col6a1−/− mice are outlined in the Table. Col6a1−/− mice were consistently smaller than WT mice, and heart weights were lower in Col6a1−/− mice in sham and MI groups when compared to WT. After MI the ejection fraction, fractional shortening and cardiac index were higher in Col6a1−/− hearts, whereas left ventricular (LV) mass and wall thinning were reduced.
Myocardial Integrity and Cardiac Function Are Significantly Preserved in Col6a1−/− Mice
Whole heart images taken 8 weeks after MI demonstrate preserved LV wall integrity in the Col6a1−/− hearts (Figure 1A). Gross observation and measurement of 2-dimensional guided M-mode tracings show improved wall thickness, chamber dimension, and anterior wall kinesis in Col6a1−/− MI mice (Figure 1B), illustrating the preserved myocardial function in the knockout hearts. Area at risk percentage (Supplemental Figure I) and infarct/LV area ratios were not significantly different in the Col6a1−/− mice versus WT 3 days after MI; however, these ratios were diminished in the knockouts compared to WT at 8 weeks after MI (0.25±0.01 versus 0.37±0.05, respectively; Figure 1C, D).
Reduced Collagen Volume and Decreased Long-Term Myocyte/Nonmyocyte Apoptosis in the Col6a1−/− Hearts
Quantitative analysis of total collagen content in the LV and infarcted zones was visualized by Masson trichrome and picrosirius red staining (Figure 2A). Collagen volume (percent collagen/LV area) was significantly reduced in Col6a1−/− MI mice compared to WT (15.89±0.84 versus 29.42±4.05; Figure 2B; P<0.05). Collagen levels of WT and Col6a1−/− sham mice were not significantly different (data not shown).
TUNEL staining revealed an initial increase in apoptosis within the infarcted area of the Col6a1−/− hearts at 3 days (acute phase), followed by a reduction by 14 days (chronic phase) relative to WT hearts (Figure 2C, D; P<0.05). Importantly, the ratio of myocyte/nonmyocyte apoptosis by day 14 was significantly decreased in the knockout hearts compared to WT (Figure 2E).
Improved Cardiac Function and LV Dimensions in Col6a1−/− Mice 10 Days to 8 Weeks After MI
Cardiac function was assessed using 2-dimensional and Doppler echocardiography on mice 3 days to 8 weeks after MI. Echocardiography at 3 days (Figure 3A) revealed that cardiac function is not significantly different between the null and WT MI mice. Differences in LV diastolic volume were also not apparent at this early time (Figure 3B). However, serial measurements of function and remodeling revealed that in Col6a1−/− mice, ejection fraction was preserved as early as 3 weeks and persisted to 8 weeks after MI (Figure 3C, E; 43.9%±3.3% versus 29.1%±4.3%; P<0.05) and LV chamber volume was reduced beginning at 10 days after MI (Figure 3D, F; 25.8%±7.9% versus 62.6%±16.5% increase in LV volume over shams; P<0.05). Cardiac index increased in Col6a1−/− mice after MI versus WT (0.55±0.02 versus 0.48±0.04; P<0.05; Table).
These data are the first to demonstrate that the lack of collagen VI significantly and paradoxically improves remodeling after MI in response to permanent left anterior descending artery occlusion. We originally predicted that the collagen VI-deficient mice would experience deficits in remodeling, because this collagen has been proposed to play a critical role in organizing and anchoring the fibrillar type I and III collagen network.9,17 This, along with the information taken from other tissues (skin, tendon, skeletal muscle), and collagen knockouts have all shaped our original hypothesis that deletion of collagen VI would result in a loss of function.
Elegant electron microscopy studies uncovered the function of collagen VI to organize the fibrillar collagen (mainly type I) network in skin.9 Izu et al16 recently reported significantly decreased collagen diameter and compromised mechanical properties in tendons of the Col6a1−/− mice. Another intriguing study demonstrated that collagen knockout null mice with experimentally induced hypertension had a poorly organized fibrillar collagen network, impaired microvascular hemodynamics, and irregularly organized cardiac myocytes.18 Our results are particularly “paradoxical” because of the known skeletal muscle phenotype of this knockout model originally reported as a model of Bethlem Myopathy by Bonaldo et al.10 This seminal study was followed by several subsequent reports indicating that the skeletal myocytes had premature mitochondrial transition pore opening and apoptosis leading to skeletal muscle dystrophy as the mice aged beyond 32 weeks.12 Prevention of mitochondrial transitional pore opening has been shown to be cardioprotective,19 indicating that this may be a common target for prevention of skeletal and cardiac myocyte dysfunction and death. It is important to note in our study that we utilized young mice for our MI studies (12–16 weeks), an age at which there were no differences in baseline cardiac function and no outward signs of skeletal muscle weakness. It is of interest to perform aging studies on the Col6a1−/− mice to determine if the cardioprotection seen in the current study persists after the onset of Bethlam myopathy.
The improvement in remodeling after MI in the Col6a1−/− hearts is, at least in part, likely because of accelerated remodeling and apoptosis followed by a reduction in chronic apoptosis. The increased apoptosis at 3 days after MI in the knockouts suggests that injury responses began earlier in these animals, which appears to be beneficial to the long-term outcomes. Our data also indicate that less myocyte apoptosis occurs in the knockouts after 14 days, which supports the notion that wound healing occurs, and is completed, earlier in the Col6a1−/− mice. Furthermore, the more chronic apoptosis evident at 14 days post-MI in the WT mice may contribute to the increased scarring and fibrosis, followed by long-term loss of cardiac function. Other cardioprotective mechanisms may exist as well, because the structure–function relationships relating to collagen VI and the fibrillar collagens have not been established in the myocardium. The previous studies of skin9 and tendon16 (discussed) suggest that the absence of collagen VI affects fibrillar collagen network organization, which, in our case, could be creating a more biomechanically advantageous environment for wound healing after MI. Alternatively, the possibility exists that cardioprotection may involve changes in mitochondrial function;19 however, mitochondrial function is altered and induces apoptosis in the skeletal muscle of Col6a1−/− mice.12 The mechanisms responsible for these contrasting phenotypes in cardiac and skeletal muscle are not known and require further investigation.
In conclusion, the confluence of previous reports and our current findings demonstrate a critical and novel role for type VI collagen in myocardial injury and remodeling. Importantly, our study is the first to describe the cardioprotective effects of collagen VI deletion to enhance cardiac function and limit aberrant post-MI remodeling, and it identifies a potentially novel target to treat postischemic injury in the myocardium.
Sources of Funding
Supported by NIH HL-079969 and Ohio Board of Regents Grant (J.G.M.).
The authors express our appreciation to the NEOMED Department of Integrative Medical Sciences Cardiovascular Focus Group for their expert critiques and suggestions, and to Dr Vahagn Ohanyan and the Echocardiography Core for their expert assistance.
In January 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.88 days.
This manuscript was sent to Gerd Heusch, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.111.252734/-/DC1.
Non-standard Abbreviations and Acronyms
- acute myocardial infarct
- ejection fraction
- myocardial infarction
- picrosirius red
- 2,3,5-triphenyltetrazolium chloride
- Received July 15, 2011.
- Revision received February 9, 2012.
- Accepted February 9, 2012.
- © 2012 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Collagen VI organizes and anchors the fibrillar collagen network in many tissues, and its deficiency causes age-related defects in skeletal muscle function.
Postinfarction remodeling depends on collagen and extracellular matrix synthesis to stabilize the scar and improve long-term remodeling.
The prevailing idea is that deposition of fibrillar collagens type I and III are increased after myocardial infarction (MI) and are the key mediators of infarct scar assembly.
Type VI collagen deposition increases after MI; however, the importance of this nonfibrillar collagen in remodeling after MI is unknown.
What New Information Does This Article Contribute?
Knockout of type VI collagen improves post-MI remodeling by limiting infarct size, collagen deposition, and chronic myocyte apoptosis.
Absence of collagen VI preserves long-term cardiac performance after MI, prevents left ventricular (LV) wall thinning and limits LV chamber dilation.
The beneficial effects of collagen VI deletion suggest that this nonfibrillar collagen plays a key role in post-MI wound healing and remodeling.
Type VI collagen is a nonfibrillar filamentous collagen produced by activated fibroblasts that plays roles in fibrillogenesis and organization and anchoring of fibrillar collagens. Our goal was to determine whether collagen VI contributes significantly to wound healing after MI by using a relevant in vivo MI model (collagen VI-deficient mice). This study is the first to our knowledge to report an unexpected and novel cardioprotective effect of collagen VI deletion in preserving cardiac structure and preventing pathological remodeling after MI. These findings may provide the basis for the development of collagen-based therapies to limit adverse remodeling after MI.